Nucleic acid modulators of glycoprotein VI

ABSTRACT

The present invention relates, in general, to a pharmacologic system to modulate the biology of platelets based upon a nucleic acid ligand that can interact with and modulate the activity of platelet glycoprotein GPVI to regulate platelet function. These nucleic acid ligands are also actively reversible using a modulator that inhibits the activity of the nucleic acid ligand to neutralize this pharmacologic effect and thereby restore GPVI function, including collagen binding, platelet adhesion, collagen-induced platelet activation, and collagen-induced platelet aggregation. The invention further relates to compositions comprising the nucleic acid ligand, the ligand and a modulator, methods to generate the nucleic acid ligand and its modulator, as well as methods of using these agents and compositions in medical therapeutic and diagnostic procedures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 12/793,572 filed onJun. 3, 2010, now U.S. Pat. No. 8,318,923, which claims priority to U.S.Provisional Application No. 61/183,847, filed Jun. 3, 2009 and U.S.Provisional Application No. 61/300,951, filed Feb. 3, 2010, the contentsof which are hereby incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

A “Sequence Listing” has been submitted with this application in theform of a text file, created Feb. 28, 2012, named““108158009US02seq.txt” (33568 bytes), the contents of which areincorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates, in general, to an anti-plateletpharmacologic system comprising a nucleic acid ligand that binds to andregulates the activity of the platelet-specific protein glycoprotein VI(GPVI). These nucleic acid ligands are also actively reversible using amodulator that inhibits the activity of the nucleic acid ligand toneutralize its pharmacologic effect and thereby restore GPVI function.The invention further relates to compositions comprising the nucleicacid ligand and/or a modulator as well as methods of using these agentsand compositions in treating platelet-mediated diseases and disorders.

BACKGROUND

Platelets are small, anuclear blood cells which are fairly quiescentunder normal conditions but which respond immediately to vascular injuryby adhesion, activation, aggregation, and thrombus formation. Theprimary function of platelets is to stop blood loss after tissue traumaand exposure of the subendothelial matrix. It is well known that damageto a blood vessel can expose extracellular matrix components to theblood, particularly von Willebrand factor (VWF), collagen, fibronectin,thrombospondin, and laminin. Interaction of platelets with these exposedmolecules results in activation of the platelet cells.

While platelets have long been recognized as having a predominant rolein hemostasis and thrombosis, it is becoming increasingly recognizedthat platelets may also play a significant role in a variety of otherdisorders such as inflammation, tumor growth and metastasis, andimmunological host defense. Accordingly, platelet receptor proteins areattractive targets for regulation of platelet function as a means oftreating or preventing platelet-mediated disorders.

Glycoprotein VI (GPVI) is a particularly attractive target as it is atransmembrane protein specifically expressed on platelets. Cell-specificmolecules having extracellular exposure offer both accessibility to atherapeutic moiety and the potential for minimal—if any—adverse sideeffects, due to the limited expression profile.

Glycoprotein VI (GPVI) is a major platelet signaling receptor forcollagen shown to have a role in generating intracellular signals thatmediate platelet activation (Watson et al. Platelets, 2000, 11:252-258).GPVI is a 62-65 kDa glycoprotein in the Ig superfamily consisting of twoIg C2 loops that contain a collagen binding domain, representingpotential drug target sites. The protein is very highly glycosylated andsialylated in vivo, and a single glycosylation site is found on theouter Ig-C2-like domain. As a member of the immunoglobulin superfamily,GPVI is related to natural killer receptors. Its signaling may bemediated indirectly through the γ-chain of FcR or directly through theGPVI cytoplasmic domain. The FcR γ-chain contains an immunoreceptortyrosine-based activation motif and in conjunction with the non-tyrosinekinase SYK and the adapters LAT and LCP2 leads to the activation ofphospholipase C2 and associated intracellular signaling pathways(Lankhof et al. Thromb Haemost, 1996, 75:950-958).

The importance of GPVI in the physiology and clinical events associatedwith clot formation has been recognized and is supported byepidemiologic associations of GPVI levels with onset of acute coronarysyndrome (ACS) and stroke events (Bigalke et al. American Heart Journal,2008, 156:193-200; Bigalke et al., European Journal of Neurology, 2009,101:911-915), and the resistance to thrombosis demonstrated in GPVIdeficient mice (Denis et al. Arteriosclerosis, Thrombosis and VascularBiology, 2007, 27:728-739). Further, Gawaz et al have shown thatradiolabeled GPVI could be used in scintillographic imaging of vascularlesions in mice as it binds specifically to the injured region,indicating that collagen is exposed at these sites (Thromb Haemost.,2005, 93:910-913). Penz et al. (FASEB J. 2005, 19:898-909) have shownthat human atheromatous plaques from patients with carotid stenosiscontained collagen type I and type III structures that were able toactivate platelets. Further, blockage or absence of GPVI was able toprevent thrombus formation, whereas blockage of α₂β₁ had little effect.Similarly blocking collagen with anti-collagen antibodies or degradingit with collagenase prevented thrombus formation.

More recently, GPVI has been linked to a broader role in disordersassociated with platelet malfunctioning and abnormal collagenexpression. This is due in part to the fact that platelets function bothin adhesion following vascular injury as in the case of thrombosis, andin the release of a variety of inflammatory mediators and cytokines.Moreover, following reaction of platelets with a site of vascularinjury, subsequent activation of the platelets results in release ofcytokines and other regulatory molecules. Thus, although it may seemthat the disorders associated with abnormal activation of platelets arediverse, these disorders are linked by their dependence on plateletfunction. Accordingly, inhibiting or preventing activation of plateletscan provide valuable therapeutic effects.

Platelet-mediated disorders include vascular diseases as well as avariety of disorders associated with high-risk diabetes. Inflammatorydisorders shown to be platelet-mediated include inflammatory arthritidesand scleroderma. The role of inflammation and white blood cell activityhas long been known in inflammatory joint disease. More recently, thepresence of platelets in synovial fluid of inflamed joints has beenidentified (Boilard et al., Science, 2010, 327:580-583). Moreover,platelet microparticles in joint fluid from patients suffering frominflammatory arthritis have been shown to be proinflammatory, elicitingcytokine responses from synovial fibroblasts via IL-1. Bothpharmacologic and genetic approaches showed that GPVI plays a key rolein this proinflammatory nature of platelets in arthritis.

Other disorders shown to be associated with GPVI expression on thesurface of platelets include experimental tumor metastasis (Jain et al.,J. Thromb Haemostasis, 2009, 7:1713-1717), diabetes (Cabeza et al.,2004, 53:2117-2121) and infection by Hepatitis C virus (Zahn et al.,Diabetes, 2004, 53:2117-2121).

Despite the expanding role GPVI has been shown to have in plateletfunction and related physiological maladies, efforts to discover anddevelop antagonists of this receptor have been limited. Active control,or modulation, of the intensity and duration of antiplatelet therapy canprovide significant clinical benefit. Therefore, there remains a need inthe art for modulatable agents designed to specifically target andregulate the function of the GPVI protein.

SUMMARY OF THE INVENTION

Described herein are compositions directed to nucleic acid ligands whichspecifically bind glycoprotein VI (GPVI), methods and treatments of useof nucleic acid ligands which specifically bind glycoprotein VI (GPVI),and modulators thereof.

In one aspect, a GPVI ligand is provided, wherein the ligand comprisesan isolated nucleic acid sequence. In another embodiment, at least onenucleotide is a ribonucleotide. In another embodiment, at least onenucleotide is a deoxyribonucleic acid. In still another embodiment, theisolated nucleic acid sequence GPVI ligand comprises a mixture ofribonucleotides and deoxyribonucleotides.

In one embodiment, the nucleic acid GPVI ligand comprises a secondarystructure comprising one, two, or three stems and one, two, three, orfour, loops.

In one embodiment, the GPVI ligand comprises a secondary structure,wherein the secondary structure comprises, in a 5′ to 3′ direction, afirst stem region, a first loop region, a second stem region, a secondloop region, a third loop region, a third stem region and a fourth loopregion. In another embodiment, the GPVI ligand consists essentially of,in a 5′ to 3′ direction, a first stem region, a first loop region, asecond stem region, a second loop region, a third loop region, a thirdstem region and a fourth loop region.

In one embodiment, the nucleic acid GPVI ligand secondary structure isconfigured in the 5′-3′ direction as the first stem, the first loop, thesecond stem connected to the second loop and to the third loop, a thirdstem connected to the third loop and to the fourth loop, and the fourthloop. In one embodiment, the second stem is connected to the first, thesecond, and the third loops.

In one embodiment, the fourth loop of the nucleic acid GPVI ligandcomprises a first consensus sequence consisting of UAA. In anotherembodiment, the fourth loop comprises the sequence consisting of(G/A)UAA.

In one embodiment, the third stem comprises the sequence consisting of aG-C basepair followed by a C-G basepair.

In one embodiment, the nucleic acid GPVI ligand comprises GC(G/A)UAAGC.In another embodiment, the third stem and the fourth loop of the nucleicacid ligand comprise GC(G/A)UAAGC. In yet another embodiment, the thirdstem and the fourth loop of the nucleic acid ligand comprises a sequenceselected from the group consisting of GCAUAAGC and GCGUAAGC.

In one embodiment, the third loop comprises the sequence YD, wherein Yis a pyrimidine and D is not cytosine. In another embodiment, the thirdloop comprises the sequence YU, YG or YA. In still another embodiment,the third loop comprises the sequence UU, UG, UA, CU, CG or CA. Inanother embodiment, the third loop consists of the sequence YD.

In one embodiment, the first loop comprises the sequence GAC.

In one embodiment, the isolated nucleic acid GPVI ligand sequence isabout 20 nucleotides (nt) to about 50 nt in length, about 20 nt to about45 nt in length, about 20 nt to about 40 nt in length, about 20 nt toabout 35 nt in length, about 20 nt to about 30 nt in length, or about 30nt to about 35 nt in length.

In one embodiment, the GPVI ligand comprises an isolated nucleic acidsequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:14 and SEQ ID NO:15. In anotherembodiment, the GPVI ligand comprises an isolated nucleic acid sequenceselected from the group consisting of SEQ ID NO:28 through SEQ ID NO:33,inclusive. In still another embodiment, the GPVI ligand comprises anisolated RNA nucleic acid sequence selected from the group consisting ofEF-1-RNA, EF-2-RNA, EF-3-RNA, E2-6-RNA, EF-22-RNA and EF-31-RNA. Inanother embodiment, the GPVI ligand comprises an isolated RNA nucleicacid sequence selected from the group consisting of EF-1-modified,EF-2-modified, EF-3-modified, E2-6-modified, EF-22-modified andEF-31-modified. In another embodiment, the GPVI ligand comprises anisolated nucleic acid sequence which is at least 80% identical to thesequence selected from the group consisting of SEQ ID NO:28 through SEQID NO:33, inclusive.

In one embodiment, the GPVI ligand is selected from the group consistingof RB424, RB426, RB427, RB428, RB429, RB430, RB445, RB446, RB447, RB431,RB432, RB433, RB434, RB435, RB436, RB439, RB440, RB441, RB442, RB443 andRB444.

In one embodiment, the GPVI ligand is selected from the group consistingof RB448, RB452, RB453, RB455, RB460, RB462, RB466, RB478, RB480, RB488,EB490, RB491, RB492, RB493, RB495, RB496, RB497, RB498, RB499, RB500,RB502, RB503, RB504, RB505, RB506, RB507, RB508, RB517, RB518, RB519,RB520, RB521, RB522, RB523, RB524, RB525, RB526, RB527, RB528, RB531,RB532, RB533, RB534, RB535, RB536, RB537, RB538, RB540, RB541, RB542,RB546, RB547, RB548, RB549, RB550, RB551, RB552, RB553, RB554, RB555,RB556, RB560, RB561, RB566, RB567, RB569, RB570, RB571.

In one embodiment, the isolated nucleic acid sequence of the GPVI ligandcomprises one or more ribonucleotides, deoxyribonucleotides, or amixture both ribonucleotides and deoxyribonucleotides.

In one embodiment, one or more of the nucleotides of the isolatednucleic acid GPVI ligand sequence is modified. In another embodiment,the one or more nucleotides comprise a modification at the 2′ hydroxylposition. In another embodiment, the modification is selected from thegroup consisting of 2′-O-methyl and 2′-fluoro. In yet anotherembodiment, the one or more nucleotides is 2′-O-methyl cytosine,2′-O-methyl uridine, 2′-O-methyl adenosine or 2′-O-methyl guanosine. Instill another embodiment, the one or more nucleotides is a 2′ fluorocytidine or a 2′ fluoro uridine.

In one embodiment, the one or more nucleotides comprising a modificationis selected from the group consisting of 5-fluorouracil,5-fluorocytosine, 5-bromouracil, 5-bromocytosine, 5-chlorouracil,5-chlorocytosine, 5-iodouracil, 5-iodocytosine, 5-methylcytosine,5-methyluracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylamin-O-methylthiouridine, 5-carboxymethylamin-O-methyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 6-methylcytosine, N6-adeninc,7-methylguanine, 5-methylamin-O-methyluracil,5-methoxyamin-O-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 5-methoxycytosine,2-methylthio-N6-isopentenyladenine, uracil oxyacetic acid (v),butoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil oxyacetic acid (v), 5-methyl thiouracil,3-(3-amino-3-N carboxypropyl)uridine, (acp3U), and 2,6-diaminopurine.

In one embodiment, the GPVI ligand comprises at least one modified sugarmoiety.

In one embodiment, the GPVI ligand comprises at least one modifiedphosphate backbone.

In one embodiment, the isolated nucleic acid GPVI ligand sequencecomprises an inverted thymine at its 3′ end.

In one embodiment, the nucleic acid GPVI ligand comprises a spacer. Inanother embodiment, the spacer is a glycol spacer. In anotherembodiment, the second loop of the nucleic acid GPVI ligand comprisesthe glycol spacer. In yet another embodiment, the second loop of thenucleic acid GPVI ligand consists of a glycol spacer. In still anotherembodiment, the glycol spacer is provided by incorporation of9-O-Dimethoxytrityl-triethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. In yetanother embodiment, the glycol spacer is attached to the 3′ end of afirst internal nucleotide of the isolated nucleotide GPVI ligandsequence and is attached to the 5′ end of a second internal nucleotideadjacent to the first internal nucleotide of the isolated nucleotideGPVI ligand sequence.

In one embodiment, the nucleic acid GPVI ligand comprises an aliphaticamino linker. In another embodiment, the aliphatic amino linker isattached to the 5′ end of the isolated nucleic acid GPVI ligandsequence. In yet another embodiment, the aliphatic amino linker isattached to the 3′ end of the isolated nucleic acid GPVI ligandsequence. In still another embodiment, the aliphatic amino linker isprovided by incorporation of 6-(trifluoroacetamino)hexanol(2-cyanoethyl-N,N-diisopropyl)phosphoramidite.

In one embodiment, the isolated nucleic acid GPVI ligand is linked to atleast one hydrophilic moiety. In another embodiment, the at least onehydrophilic moiety is a polyalkylene glycol.

In one embodiment, the GPVI ligand comprises a polyalkylene moietyattached to the 5′ end and/or the 3′ end of the isolated nucleic acidsequence. In another embodiment, the polyalkylene moiety is attached viaa linker. In yet another embodiment, the linker is an aliphatic aminolinker.

In one embodiment, the GPVI ligand is linked to a 40 KD polyethyleneglycol (PEG) moiety using a six carbon amino linker. In a anotherembodiment, the six carbon amino linker is attached to the PEG moietythrough an amide attachment. In a yet another embodiment, the PEG moietyis two twenty KD PEG moieties which are attached to one or more aminoacids, such as lysine, which is attached via an amide bond to the sixcarbon amino linker.

In one embodiment, the first nucleic acid GPVI ligand comprises aphosphorothioate linkage.

In one embodiment, the nucleic acid GPVI ligand specifically binds toGPVI (SEQ ID NO:1). In another embodiment, the nucleic acid GPVI ligandspecifically binds to the extracellular domain of GPVI (SEQ ID NO:2).

In one embodiment, the GPVI ligand has a dissociation constant of about20 nanomolar (nM) or less.

In one embodiment, the GPVI ligand has a dissociation constant whichranges from about 400 picomolar (pM) to about 10 nM.

In one embodiment, the GPVI ligand has a dissociation constant whichranges from about 100 pM to about 10 nM.

In one embodiment, the nucleic acid GPVI ligand inhibits binding of GPVIto collagen. In another embodiment, the nucleic acid GPVI ligandinhibits intracellular signaling via GPVI. In another embodiment, theinhibiting of intracellular signaling via GPVI using a GPVI ligandcomprises reducing generation of inositol trisphosphate or inhibitingfluctuations in intracellular calcium levels.

In one embodiment, the nucleic acid GPVI ligand inhibits binding of GPVIto collagen-related peptide (CRP) and/or to convulxin.

In one embodiment, the nucleic acid GPVI ligand inhibits binding of GPVIto both collagen and to CRP.

In one embodiment, the nucleic acid GPVI ligand inhibits binding of GPVIto collagen but does not inhibit binding of GPVI to CRP. In anotherembodiment, the nucleic acid GPVI ligand inhibits binding of GPVI toboth collagen and CRP but does not inhibit binding of GPVI to convulxin.

In one embodiment, binding of the nucleic acid GPVI ligand to GPVIstabilizes an active conformation of GPVI. In another embodiment,binding of the nucleic acid GPVI ligand to GPVI stabilizes an inactiveconformation of GPVI. In yet another embodiment, binding of the nucleicacid GPVI ligand to GPVI inhibits interaction between GPVI and the FcR γsubunit.

In another embodiment, binding of the GPVI ligand to GPVI results ininhibition of, or reduction of, GPVI activity. In yet anotherembodiment, binding of the GPVI ligand to GPVI results in the inabilityof, or the reduction in ability of, GPVI to interact with FcR γ-chain.In still another embodiment, binding of the GPVI ligand to GPVIexpressed on the surface of a platelet results in an inhibition of, orreduction of, platelet adhesion. In still another embodiment, binding ofthe GPVI ligand to GPVI expressed on the surface of a platelet resultsin an inhibition of, or reduction of, platelet activation. In stillanother embodiment, binding of the GPVI ligand to GPVI expressed on thesurface of a platelet results in an inhibition of, or reduction of,platelet aggregation.

In one embodiment, the GPVI ligand binds to and decreases or inhibits afunction of a variant of GPVI, wherein said GPVI variant is at least80%, 85%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQID NO:1.

In one embodiment the GPVI ligand has a dissociation constant (“K_(d)”)for GPVI of less than about 100 micromolar (μM), less than about 1 μM,less than about 500 nanomolar (nM), less than about 100 nM, less thanabout 50 nM, less than about 1 nM, less than about 500 picomolar (pM),less than about 300 pM, less than about 250 pM, or less than about 200about pM.

In a second aspect, a modulator to the GPVI ligand is provided, whereinthe modulator reverses, partially or completely, the activity of a GPVIligand.

In one embodiment, the modulator comprises an isolated nucleic acidsequence. In another embodiment, the modulator comprises a DNA sequence,an RNA sequence, a polypeptidc sequence, or any combination thereof.

In one embodiment, the modulator of a GPVI nucleic acid ligand isselected from the group consisting of a ribozyme, a DNAzyme, a peptidenucleic acid (PNA), a morpholino nucleic acid (MNA), and a lockednucleic acid (LNA).

In one embodiment, the modulator of a GPVI nucleic acid ligand isselected from the group consisting of a ribozyme, a DNAzyme, a peptidenucleic acid (PNA), a morpholino nucleic acid (MNA), and a lockednucleic acid (LNA), wherein the modulator specifically binds to orinteracts with at least a portion of a GPVI nucleic acid ligand.

In one embodiment, the modulator is selected from the group consistingof a nucleic acid binding protein or peptide, a small molecule, anoligosaccharide, a nucleic acid binding lipid, a polymer, ananoparticle, and a microsphere, wherein the modulator binds to orinteracts with at least a portion of a GPVI nucleic acid ligand.

In one embodiment, the modulator is a nucleic acid modulator comprisingdeoxyribonucleotides, ribonucleotides, or a mixture ofdeoxyribonucleotides and ribonucleotides. In another embodiment thenucleic acid modulator comprises at least one modifieddeoxyribonucleotide and/or at least one modified ribonucleotide.

In one embodiment, the modulator is an oligonucleotide which iscomplementary to at least a portion of the GPVI nucleic acid ligand. Inanother embodiment, the modulator is an oligonucleotide which iscomplementary to at least a portion of a loop in the GPVI ligand. Inanother embodiment, the modulator is an oligonucleotide complementary toat least the first loop, the second stem, and the second loop of thenucleic acid GPVI ligand. In another embodiment, the modulator is anoligonucleotide complementary to the third loop, the third stem, thefourth loop, and the first stem of the GPVI ligand.

In one embodiment, the modulator comprises an isolated nucleic acidsequence, wherein the sequence is about 10 nt to about 30 nt, about 10nt to about 25 nt, about 10 nt to about 20 nt, about 10 nt to about 15nt, or about 15 nt to about 20 nt in length.

In one embodiment, one or more of the nucleotides of the nucleic acidmodulator sequence is modified. In another embodiment, the one or morenucleotides comprise a modification at the 2′ hydroxyl position. Inanother embodiment, the modification is selected from the groupconsisting of 2′-O-methyl and 2′-fluoro. In yet another embodiment, theone or more nucleotides is 2′-O-methyl cytosine, 2′-O-methyl uridine,2′-O-methyl adenosine, 2′-O-methyl guanosine or a 2′-O-methyl thymidine.In still another embodiment, the one or more nucleotides is a 2′ fluorocytidine, a 2′ fluoro uridine, a 2′ fluoro adenosine or a 2′-fluoroguanosine.

In one embodiment, the modification of one or more nucleotides of thenucleic acid modulator comprises a modification selected from the groupconsisting of 5-fluorouracil, 5-fluorocytosinc, 5-bromouracil,5-bromocytosinc, 5-chlorouracil, 5-chlorocytosinc, 5-iodouracil,5-iodocytosine, 5-methylcytosine, 5-methyluracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylamin-O-methyl thiouridine,5-carboxymethylamin-O-methyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 6-methylcytosine, N6-adenine,7-methylguanine, 5-methylamin-O-methyluracil,5-methoxyamin-O-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 5-methoxycytosine,2-methylthio-N6-isopentenyladenine, uracil oxyacetic acid (v),butoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil oxyacetic acid (v), 5-methyl thiouracil,3-(3-amino-3-N carboxypropyl)uridine (acp3U), and 2,6-diaminopurine.

In one embodiment, the modulator comprises as least one modified sugarmoiety.

In one embodiment, the modulator comprises at least one modifiedphosphate backbone.

In one embodiment, the modulator comprises an oligonucleotide whichhybridizes at physiological conditions to the fourth loop of the GPVIligand. In another embodiment, the oligonucleotide modulator comprisesthe sequence 3′-AUU-5′.

In one embodiment, the modulator comprises an oligonucleotide whichhybridizes under physiological conditions to the first loop of the GPVIligand. In another embodiment, the oligonucleotide modulator comprisesthe sequence 3′CUG-5′.

In one embodiment, the modulator comprises the sequence selected fromthe group consisting of SEQ ID NOs:74-88, inclusive. In anotherembodiment, the modulator is selected from the group consisting ofRB416, RB417, RB418, RB419, RB420, RB421, RB422, RB423, RB513, RB514,RB515, RB516, RB543, RB544, and RB545.

In one embodiment, the modulator disrupts the secondary structure of thenucleic acid GPVI ligand. In another embodiment, the modulatorstabilizes the secondary structure of the GPVI ligand.

In one embodiment, the modulator disrupts the tertiary structure of thenucleic acid GPVI ligand. In another embodiment, the modulatorstabilizes the secondary structure of the GPVI ligand.

In one embodiment, the binding of the modulator to the GPVI ligandexposes a suicide position within the GPVI ligand, thereby disruptingthe secondary structure of the GPVI ligand and leading to enhanceddestruction of the nucleic acid GPVI ligand by nucleases.

In one embodiment, binding of the modulator to a GPVI ligand-GPVIcomplex reduces or eliminates binding of the GPVI ligand to GPVI.

In another aspect, a method of modulating the activity of a GPVI ligandis provided.

In one embodiment, a method of modulating the activity of a nucleic acidligand to GPVI by administering a modulator of the GPVI ligand to a hostwho has been administered the nucleic acid GPVI ligand is provided. Inone embodiment, the modulator can be a oligonucleotide modulator, orderivative thereof, and in certain embodiments, is complimentary to aportion of the nucleic acid GPVI ligand.

In a further aspect, a method of regulating GPVI function using a GPVIligand is provided.

In one embodiment, the method for regulating GPVI function comprisesadministering to a host a therapeutically effective amount of a GPVIligand. In another embodiment, the method further comprisesadministering a GPVI ligand modulator to the host.

In another aspect, a method of treating or ameliorating aplatelet-mediated disease or disorder is provided.

In one embodiment, the method comprises administering to a host in needthereof a therapeutically effective dose of a GPVI ligand that binds toGPVI. In another embodiment, the host is diagnosed with high-riskdiabetes. In still another embodiment, the host is diagnosed with acancer at high risk of metastasis.

In one embodiment, the platelet-mediated disease or disorder is selectedfrom the group consisting of cerebrovascular disorders, acute coronarysyndromes, diabetes-related disorders, autoimmune inflammatorydisorders, and cancer.

In one embodiment, the cerebrovascular disorder is a thrombosis,thromboembolism, or transient ischemia attack (TIA). In anotherembodiment, the acute coronary syndrome is due to coronary thrombosis,unstable angina or myocardial infarction. In still another embodiment,the diabetes-related disorder is diabetic retinopathy, diabeticvasculopathy, atherosclerosis, ischemic stroke, peripheral vasculardisease, acute renal injury or chronic renal failure. In anotherembodiment, the autoimmune inflammatory disorder is scleroderma,rheumatoid arthritis, or an inflammatory autoimmune disorder selectedfrom the group consisting of psoriatic arthritis, reactive arthritis,inflammatory bowel disease and ankylosing spondylitis. In oneembodiment, the cancer is selected from lung cancer, breast cancer,prostate cancer, pancreatic cancer, brain cancer, bone cancer and livercancer.

In one embodiment, the GPVI ligand is administered by parenteraladministration, intravenous injection, intradermal delivery,intra-articular delivery, intra-synovial delivery, intrathecal,intra-arterial delivery, intracardiac delivery, intramuscular delivery,subcutaneous delivery, intraorbital delivery, intracapsular delivery,intraspinal delivery, intrasternal delivery, topical delivery,transdermal patch delivery, rectal delivery, delivery via vaginal orurethral suppository, peritoneal delivery, percutaneous delivery,delivery via nasal spray, delivery via surgical implant, delivery viainternal surgical paint, delivery via infusion pump or delivery viacatheter.

In another aspect, a method for treating a host in need thereof byadministering a GPVI ligand, wherein the GPVI ligand regulates plateletfunction is provided.

In one embodiment, a therapeutically effective dose of GPVI isadministered.

In one embodiment, the therapeutically effective dose reduces orinhibits platelet adhesion and/or aggregation.

In one aspect, a pharmaceutical composition comprising a therapeuticallyeffective amount of a nucleic acid GPVI ligand which binds GPVI isprovided.

In one aspect, a pharmaceutical composition comprising a therapeuticallyeffective amount of a modulator, wherein the modulator regulates theactivity of a nucleic acid GPVI ligand which binds GPVI, is provided.

In one embodiment, the pharmaceutical composition comprises a GPVIligand and pharmaceutically-acceptable excipients. In anotherembodiment, the pharmaceutical composition is a liquid suitable forintravenous injection. In yet another embodiment, the pharmaceuticalcomposition is a liquid or dispersion suitable for subcutaneousinjection.

In one aspect, a kit comprising a therapeutically effective amount of aGPVI nucleic acid ligand and/or a modulator which regulates the activityof the GPVI nucleic acid ligand is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the SELEX nucleic acid ligand selection process.

FIG. 2 shows the selection conditions for the SELEX rounds performed toidentify nucleic acid ligands to GPVI.

FIG. 3 shows the binding curves of trace ³²P end-labeled librariesenriched in nucleic acid ligands to GPVI.

FIGS. 4A-B shows a primary random region derived sequence identified forindividual clones in the “E/F” selection series after Round 10 ofselection and for individual clones in the “E2” selection series afterRound 10 of selection.

FIG. 5 shows a minimal predicted primary sequence required for GPVIbinding and the predicted conserved secondary structure for sequencesidentified in the selection procedure. “L” denotes a loop, “S” denotes astem region.

FIG. 6 is a graph of the binding of the EF-3 GPVI ligand to GPVI, withor without annealing of a 3′ primer to EF-3 to mask the 3′ fixed regionof EF-3 during protein interaction.

FIG. 7 is a graph of binding curves of truncated ligands to GPVIcompared to parent GPVI ligands EF-2 and EF-3.

FIGS. 8A-B shows the sequence and predicted secondary structure ofseveral truncations of EF-3 and EF-31 GPVI ligand truncation variants.

FIG. 9 shows predicted secondary structure of the truncated sequenceEF-3 T2 and EF-2 T2 GPVI ligands and the location of inactive pointmutants.

FIGS. 10A-D shows results of GPVI binding assays for GPVI nucleic acidligand variants.

FIGS. 11A-B shows predicted secondary structure and sugar substitutionsof various GPVI nucleic acid ligands.

FIGS. 12A-D shows results of GPVI binding assays for GPVI nucleic acidligand variants.

FIGS. 13A-B illustrate a hexaethylene glycol spacer phosphoramidite andthe spacer phosphoramidite incorporated between two nucleotides in anucleic acid sequence.

FIGS. 14A-B shows PEG moieties which may be conjugated to a GPVI ligandvia a linker and a configuration of a conjugated moiety.

FIG. 15 is a graph of collagen-induced platelet aggregation expressed asa percentage of control for EF-2 and EF-3 GPVI ligand truncationvariants as compared to variants containing inactivating pointmutations.

FIGS. 16A-D is a graph of collagen-induced platelet aggregationexpressed as percentage of control for varying concentrations of selectGPVI nucleic acid ligands

FIGS. 17A-C shows graphs of CRP-induced platelet aggregation expressedas percentage of control for varying concentrations of select GPVInucleic acid ligands.

FIGS. 18A-D shows graphs of collagen-induced platelet aggregationexpressed as percentage of control for varying concentrations of selectGPVI nucleic acid ligands

FIGS. 19A-D shows graphs of CRP-induced platelet aggregation expressedas percentage of control for varying concentrations of select GPVInucleic acid ligands.

FIGS. 20A-B shows graphs of collagen and CRP-induced plateletaggregation expressed as a percentage of control at varyingconcentrations of GPVI ligand RB571 in platelet rich plasma.

FIG. 21 is a graph showing the effects of GPVI nucleic acid ligands onplatelet accumulation on a collagen-coated surface exposed to flowingwhole blood expressed as the % maximum response of an inactive controlligand.

FIGS. 22A-B shows graphs depicting specificity of GPVI ligand RB571binding to GPVI as compared to platelet receptors P2Y₁, P2Y₁₂ and PAR-1.

FIGS. 23A-B shows graphs depicting specificity of GPVI ligand RB571binding to GPVI as compared to the platelet receptor GP1bα-vWFinteraction or the platelet thromboxane A2 receptor.

FIG. 24 shows the predicted secondary structure of the truncatedsequence GPVI ligands EF2-T2 and EF3-T2 as well as regions ofcomplementarity between the ligands and the GPVI ligand modulatorsRB416-423.

FIGS. 25A-D shows graphs of collagen-induced platelet aggregationexpressed as a percentage of control for GPVI ligand EF2-T2 alone or incombination with various concentrations of different GPVI ligandmodulators.

FIGS. 26A-D shows graphs of collagen-induced platelet aggregationexpressed as a percentage of control for GPVI ligand EF3-T2 alone or incombination with various concentrations of different GPVI ligandmodulators.

FIGS. 27A-D shows graphs of collagen-induced platelet aggregationexpressed as a percentage of control for GPVI ligand RB490 alone or incombination with various concentrations of different GPVI ligandmodulators.

FIG. 28 shows a graph depicting durability of reversal of anti-GPVIactivity of a GPVI nucleic acid ligand RB490 with GPVI ligand modulatorRB515.

FIG. 29 shows predicted secondary structure of a GPVI ligand RB571 andits predicted interaction with a GPVI ligand modulator RB515.

FIGS. 30A-B shows graphs of collagen-induced and CRP-induced plateletaggregation expressed as a percentage of control for GPVI ligand RB571alone or in combination with various concentrations of GPVI ligandmodulator RB515.

FIGS. 31A-B shows graphs of collagen-induced and CRP-induced plateletaggregation expressed as a percentage of control for GPVI ligand RB571alone or in combination with GPVI ligand modulator RB515 in plateletrich plasma.

DETAILED DESCRIPTION

The present invention provides pharmaceutical compositions of nucleicacid ligands which bind to platelet membrane glycoprotein VI (GPVI),modulators of the ligands, and methods of use thereof for the treatmentof platelet-mediated diseases and disorders. Further provided arepharmaceutical formulations comprising a GPVI nucleic acid ligand and/orGPVI ligand modulator.

A. Definitions

The term “about”, as used herein when referring to a measurable valuesuch as an amount of weight, time, dose, etc. is meant to encompassvariations of ±20% or ±10%, ±5%, ±1%, or ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

A “nucleic acid ligand,” which may also referred to herein as a “ligand”or “aptamer,” is a nucleic acid that can form a tertiary structure,which allows it to interact with a target molecule. A “GPVI nucleic acidligand” or “GPVI ligand” or “anti-GPVI ligand” of “nucleic acid GPVIligand” refers to a ligand or aptamer that specifically binds to GPVI.The terms refer to oligonucleotides having specific binding regions thatare capable of forming complexes with an intended target molecule in aphysiological environment. The affinity of the binding of an ligand to atarget molecule is defined in terms of the dissociation constant (K_(d))of the interaction between the ligand and the target molecule.Typically, the K_(d) of the ligand for its target is between about 1 nMto about 100 nM. The specificity of the binding is defined in terms ofthe comparative dissociation constant of the ligand for target ascompared to the dissociation constant with respect to the ligand andother materials in the environment or unrelated molecules in general.Typically, the K_(d) for the ligand with respect to the target will be10-fold, 50-fold, 100-fold, or 200-fold less than the K_(d) with respectto the unrelated material or accompanying material in the environment.

“Ligand modulator pair” or “ligand modulator pair” is meant to include aspecified ligand to a target molecule, and a ligand modulator thatchanges the secondary and/or tertiary structure of the ligand so thatthe ligand's interaction with its target is modulated. The modulator canbe an oligonucleotide complimentary to a portion of the ligand. Themodulator can change the conformation of the ligand to reduce the targetbinding capacity of the ligand by 10% to 100%, 20% to 100%, 25%, 40%,50%, 60%, 70%, 80%, 90% or 100%, or any percentage in the range between10% and 100% under physiological conditions.

“Modulator,” “antidote,” “regulator” or “control agent” refer to anypharmaceutically acceptable agent that can bind a ligand or aptamer asdescribed herein and modify the interaction between that ligand and itstarget molecule (e.g., by modifying the structure of the ligand) in adesired manner.

“Modulate” as used herein means a lessening, an increase, or some othermeasurable change in activity.

“Pharmaceutically acceptable,” as used herein means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inhumans.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent) ofa disease state. The pharmaceutically effective dose depends on the typeof disease, the composition used, the route of administration, the typeof mammal being treated, the physical characteristics of the specificmammal under consideration, concurrent medication, and other factorsthat those skilled in the medical arts will recognize. Generally, anamount between 0.1 mg/kg and 100 mg/kg body weight/day of activeingredients is administered dependent upon the potency of the nucleicacid ligand and modulator.

A “stabilized nucleic acid molecule” refers to a nucleic acid moleculethat is less readily degraded in vivo (e.g., via an exonuclease orendonuclease) in comparison to a non-stabilized nucleic acid molecule.Stabilization can be a function of length and/or secondary structureand/or inclusion of chemical substitutions within the sugar of phosphateportions of the oligonucleotide backbone. Stabilization can be obtainedby controlling, for example, secondary structure which can stabilize amolecule. For example, if the 3′ end of a nucleic acid molecule iscomplementarily to an upstream region, that portion can fold back andform a “stem loop” structure which stabilizes the molecule.

The terms “binding affinity” and “binding activity” are meant to referto the tendency of a ligand molecule to bind or not to bind to a target.The energetics of said interactions are significant in “bindingactivity” and “binding affinity” because they define the necessaryconcentrations of interacting partners, the rates at which thesepartners are capable of associating, and the relative concentrations ofbound and free molecules in a solution. The energetics may becharacterized through, among other ways, the determination of adissociation constant, K_(d).

“Treatment” or “treating” as used herein means any treatment of diseasein a mammal, including: (a) protecting against the disease, that is,causing the clinical symptoms not to develop; (b) inhibiting thedisease, that is, arresting, ameliorating, reducing, or suppressing thedevelopment of clinical symptoms; and/or (c) relieving the disease, thatis, causing the regression of clinical symptoms. It will be understoodby those skilled in the art that in human medicine, it is not alwayspossible to distinguish between “preventing” and “suppressing” since theultimate inductive event or events may be unknown, latent, or thepatient is not ascertained until well after the occurrence of the eventor events. Therefore, as used herein the term “prophylaxis” is intendedas an element of “treatment” to encompass both “preventing” and“suppressing” as defined herein. The term “protection,” as used herein,is meant to include “prophylaxis.”

The term “effective amount” means a dosage sufficient to providetreatment for the disorder or disease state being treated. This willvary depending on the patient, the disease and the treatment beingeffected.

A GPVI nucleic acid ligand “variant” as used herein encompasses variantsthat perform essentially the same function as a GPVI nucleic acid ligandand comprises substantially the same structure.

B. Glycoprotein VI

Glycoprotein VI (GPVI) is specifically expressed on the surface ofplatelet cells. Numerous studies have shown that collagen-mediatedactivation of GPVI plays a key role in platelet adhesion andaggregation. Accordingly, GPVI is a therapeutic target of increasinginterest for the treatment of platelet and collagen-mediated diseases.

As the demarcation between physiological hemostasis and pathologicalthrombosis is very narrow, it is essential to be able to providefine-tuned control of platelet activity with respect tocollagen-mediated GPVI activation. Accordingly, also provided herein aremodulator components capable of modulating or regulating the activity ofthe disclosed GPVI ligands.

GPVI is a glycoprotein of 339 amino acid residues in length (GenBankAccession No. Q9HCN6; disclosed herein as SEQ ID NO:1). Amino acidresidues 1-20 represent a signal sequence that is cleaved to produce themature protein having 319 amino acids. GPVI has two extracellularimmunoglobulin domains, a mucin-like core, a short peptide linkersequence, a transmembrane domain and a short cytoplasmic tail that bindsFyn and Lyn Src family kinases. GPVI is also constitutively complexedwith FcR γ-chain allowing the assembly and activation of Syk andinitiating activation of a downstream signaling pathway that has manysimilarities with that employed by immune receptors. The gene encodingGPVI is found in the leukocyte receptor cluster (LRC) on humanchromosome 19. Mice that lack either GPVI or the FcRγ-chain havesignificantly impaired platelet response to collagen and reducedthrombus formation. Additionally, the Fab fragment of a novel monoclonalantihuman GPVI antibody, OM4, inhibits thrombosis in vivo in a model ofthrombosis in rats without prolongation of bleeding time seen with antiGP IIb/IIIa antibodies.

The GPVI extracellular domain (SEQ ID NO:2) has been shown tospecifically bind collagen types I-IV (Jung et al., Platelets, 2008,19:32-42). Moreover, collagen types I-IV are known to support plateletactivation, aggregation and adhesion, while the non-fibrillar collagens,types VI, VII and VIII induce only weak adhesion with no plateletaggregation. Accordingly, studies were done to identify GPVI ligandsthat bind to the GPVI extracellular domain, in order to generate apharmaceutical agent which can be useful in the treatment ofplatelet-mediated disorders or diseases.

C. Development of Nucleic Acid Ligands to GPVI

Nucleic acid ligands which specifically bind the GPVI protein wereidentified using the SELEX method. The ligands which were initiallyobtained via SELEX were then fully characterized to understand theproperties of the GPVI ligands. Such characterization includedsequencing, sequence alignment to determine conserved sequences,secondary structure prediction, and truncations and mutation analysis toidentify ligand regions most critical for the desired function ofspecifically binding and inhibiting GPVI. After identifying optimalligand sequence and secondary structures, modifications were made tooptimize the ligands for pharmaceutical use. Examples of thesemodifications include pegylation, use of a spacer within the nucleicacid ligand and selected modifications to the sugar and phosphateportion of the nucleic acid ligand. Binding assays were performed tomonitor ligand function as a result of the various modifications used.

SELEX refers to the Systematic Evolution of Ligands by EXponentialEnrichment. This method allows the in vitro evolution of nucleic acidmolecules with highly specific binding to target molecules. The SELEXmethod is described in, for example, U.S. Pat. Nos. 7,087,735;5,475,096; and 5,270,163 (see also WO 91/19813).

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievevirtually any desired criterion of binding affinity and selectivity.Starting from a mixture of nucleic acids, such as mixtures comprising asegment of randomized sequence, the SELEX method includes steps ofcontacting the mixture with the target under conditions favorable forbinding, partitioning unbound nucleic acids from those nucleic acidswhich have bound specifically to target molecules, dissociating thenucleic acid-target complexes, amplifying the nucleic acids dissociatedfrom the nucleic acid-target complexes to yield a ligand-enrichedmixture of nucleic acids, then reiterating the steps of binding,partitioning, dissociating and amplifying through as many cycles asdesired to yield highly specific, high affinity ligands to the targetmolecule.

The basic SELEX method has been modified to achieve a number of specificobjectives. For example, U.S. Pat. No. 5,707,796 describes the use ofSELEX in conjunction with gel electrophoresis to select nucleic acidmolecules with specific structural characteristics, such as bent DNA.U.S. Pat. No. 5,763,177 describes a SELEX-based method for selectingligands containing photoreactive groups capable of binding and/orphotocrosslinking to and/or photoinactivating a target molecule. U.S.Pat. No. 5,580,737 describes a method for identifying highly specificligands able to discriminate between closely related molecules, termedCounter-SELEX. U.S. Pat. Nos. 5,567,588 and 5,861,254 describeSELEX-based methods that achieve highly efficient partitioning betweenoligonucleotides having high and low affinity for a target molecule.U.S. Pat. No. 5,496,938, describes methods for obtaining improvedligands after the SELEX process has been performed. U.S. Pat. No.5,705,337, describes methods for covalently linking a ligand to itstarget.

The feasibility of identifying nucleic acid ligands to small peptides insolution was demonstrated in U.S. Pat. No. 5,648,214. The ability to useaffinity elution with a ligand to produce ligands that are targeted to aspecific site on the target molecule is exemplified in U.S. Pat. No.5,780,228, which relates to the production of high affinity ligandsbinding to certain lectins. Methods of preparing nucleic acid ligands tocertain tissues, which include groups of cell types, are described inU.S. Pat. No. 6,127,119. The production of certain modified highaffinity ligands to calf intestinal phosphatase is described in U.S.Pat. No. 6,673,553. U.S. Pat. No. 6,716,580 describes an automatedprocess of identifying nucleic acid ligands that includes the use of arobotic manipulators.

In its most basic form, the SELEX process may be defined by thefollowing series of steps:

1) A candidate mixture of nucleic acids of differing sequence isprepared. The candidate mixture generally includes regions of fixedsequences (i.e., each of the members of the candidate mixture containsthe same sequences in the same location) and regions of randomizedsequences. The fixed sequence regions are selected either: (a) to assistin the amplification steps described below, (b) to mimic a sequenceknown to bind to the target, or (c) to enhance the concentration of agiven structural arrangement of the nucleic acids in the candidatemixture. The randomized sequences can be totally randomized (i.e., theprobability of finding a base at any position being one in four) or onlypartially randomized (e.g., the probability of finding a base at anylocation can be selected at any level between 0 and 100%).

2) The candidate mixture is contacted with the selected target underconditions favorable for binding between the target and members of thecandidate mixture. Under these circumstances, the interaction betweenthe target and the nucleic acids of the candidate mixture can beconsidered as forming nucleic acid-target complexes between the targetand those nucleic acids having the strongest affinity for the target.

3) The nucleic acids with the highest affinity for the target arepartitioned from those nucleic acids with lesser affinity to the target.Because only an extremely small number of sequences (and possibly onlyone molecule of nucleic acid) corresponding to the highest affinitynucleic acids exist in the candidate mixture, it is generally desirableto set the partitioning criteria so that a significant amount of thenucleic acids in the candidate mixture (approximately 5 to 50%) areretained during partitioning.

4) Those nucleic acids selected during partitioning as having therelatively higher affinity to the target are then amplified to create anew candidate mixture that is enriched in nucleic acids having arelatively higher affinity for the target.

5) By repeating the partitioning and amplifying steps above, the newlyformed candidate mixture contains fewer and fewer weakly bindingsequences, and the average degree of affinity of the nucleic acids tothe target will generally increase. Taken to its extreme, the SELEXprocess will yield a candidate mixture containing one or a small numberof unique nucleic acids representing those nucleic acids sequences fromthe original candidate mixture which fold into a specific secondary andtertiary structure enabling the highest affinity interaction with thetarget molecule.

SELEX can be used generate bivalent binding that have two or morebinding domains with affinity for two or more epitopes of a protein,including a receptor. Specifically, in one embodiment, the process canbe used to select for nucleic acid ligands that have affinity for two ormore regions of the GPVI receptor. For example, in certain embodiments,the ligand can bind to at least two portions of the C2 regions. Incertain embodiments, the ligand affects dimerization of the GPVIreceptor, either by disrupting or stabilizing the dimeric conformation.In these embodiments, modulators can be designed to reduce binding toonly one, more than one, or all epitopes that the nucleic acid ligandbinds to. The modulator can, for example, interfere with binding to onlya single epitope, such as a C2-1 or C2-2 region of the receptor.

Nucleic acid ligands specific to GPVI may be generated by performingSELEX against short peptides which represent the extracellular domain ofthe molecule, using SELEX methods as described for example in U.S. Pat.No. 7,087,735. Alternatively nucleic acid ligands specific to GPVI canbe isolated by performing SELEX on intact platelets, platelet membranefractions enriched for the protein, on purified GPVI, or on cell-linesspecifically over-expressing the GPVI receptor using SELEX methods asdescribed, for example, in U.S. Pat. No. 6,730,482.

Additionally, the SELEX process can be directed to isolate specific GPVInucleic acid ligands using competitive affinity elution schemes, such asthose described in U.S. Pat. No. 5,780,228. For example, to isolatenucleic acid ligands specific to GPVI, elution of ligands bound to theprotein could be accomplished by addition of sufficient amounts of anactivator or binder of GPVI such as collagen, convulxin or CRP orrelated compounds.

The GPVI receptor can be a recombinantly expressed and purified proteinused for a SELEX procedure. In certain embodiments, the GPVI nucleicacid ligand binds to the GPVI receptor under physiological conditions.Physiological conditions are typically related to the level of salts andpH of a solution. In vitro, physiological conditions are typicallyreplicated in a buffer including 150 mM NaCl, 2 mM CaCl₂ 20 mM HEPES, ata pH of about 7.4. In certain embodiments, native, typicallyunactivated, platelets are used as described above to screen apopulation of nucleic acid ligands and provide an enriched population,which contains ligands directed to proteins found on platelets. Theenriched population is then used against either a stable cell lineoverexpressing the desired GPVI receptor, or a cell line that has beentransiently transfected with the protein. The secondary screening can beaccomplished either by using a modified SELEX procedure on isolatedreceptors from these cells or on the whole cells either through ligandcompetition studies or by identifying the effects on intracellularsignaling pathways.

In certain embodiments, nucleic acid ligands to specific GPVI targetscan be identified using an immobilized protein. In some of theseembodiments, a purified protein can be linked to a solid matrix by achemical linker. In other embodiments, membranes from cellsover-expressing a particular protein can be extracted using a detergent,such as an anionic detergent (e.g. cholate), to isolate a certainfraction of the proteins and the mixture coupled to an immobilizedartificial membrane. Generally, it is thought that the reorganizationcan be accomplished by removal of the detergent, during which lipids andproteins reorganize and form a layer with the hydrocarbon chains of theimmobilized artificial membrane, which is generally on a support matrixsuch as a bead.

Nucleic acid ligands isolated by these SELEX procedures specific toGPVI, which also possess a desired functional activity can be identifiedby screening nucleic acid ligands for their ability to inhibit specificagonist-induced platelet function and/or intracellular signaling eventselicited by GPVI. As the desired nucleic acid ligands are not merelybinding partners, but are inhibitors of the receptor signaling, it ispossible to identify ligands having a desired function by assessing theeffect of the ligand on platelet activity. This can includecharacterizing the effects of the ligand on various signally pathwaysknown to be regulated by GPVI. For example, GPVI signaling can lead toclustering of the GPVI protein, and to the activation of kinases tostart a local signal chain of events that activates phospholipase Cγ2,releasing the second messengers 1,4,5-inositol trisphosphates anddiacylglycerol that are responsible for raising Ca²⁺ levels andactivating protein kinase C. Any of these second messenger systems orsignals can be measured using methods well known to those havingordinary skill in the art.

In addition, collagen binding of GPVI in the presence or absence of aGPVI nucleic acid ligand, can be measured in these systems.

Ligands can also be screened for inhibition of platelet aggregation inplatelet function assays such as Light Transmittance Aggregometryperformed in platelet rich plasma and wash platelet preparations orImpedance Aggregometry performed in whole blood. Additionally, ligandscan also be screened for inhibition of platelet interaction withcollagen coated surfaces in static conditions or in flowing whole blood.The specificity of a given nucleic acid ligand for GPVI can be furtherdistinguished by the ability of the ligand to block intracellularsignaling events triggered by known agonists of the given receptor. Forexample, a nucleic acid ligand inhibitor of GPVI may be expected toblock signaling triggered by, for example, the snake C-type lectinconvulxin, collagen or collagen-related peptide (CRP). The specificityof a given nucleic acid ligand for GPVI can also be furtherdistinguished by the absence of an effect on platelet aggregation whenaggregation is triggered by an agonist that activates a receptor otherthan GPVI.

Application of any of the above-described methods, alone or incombination, will give rise to a plurality of nucleic acid ligandsspecific to GPVI. Upon identification of a nucleic acid ligand with thedesired inhibitory properties, modulators of this ligand can beidentified as described below.

D. Nucleic Acid Ligands to GPVI

The GPVI ligands disclosed herein are preferably nucleic acid ligands,such as ligands. GPVI ligands which specifically bind the GPVIextracellular domain (ECD) (amino acid residues Gln21-Lys267; SEQ IDNO:2) were selected using the SELEX method, described in more detailbelow and in Example 1, then modified to increase stability, affinityfor GPVI and/or the ability to regulate GPVI activity.

A GPVI nucleic acid ligand of the present invention is comprised of anisolated nucleic acid sequence, which can be DNA or RNA, and which canbe synthesized using modified ribo- or deoxyribonucleic acids. Asdescribed herein, if a base structure of RNA is utilized, the structurewill include uridine (U) in lieu of thymidine (T) in the base sequence.In certain embodiments described herein, the sequence of nucleic acidsis written as an RNA sequence. Similarly, in certain embodimentsdescribed herein, wherein the nucleic acid ligand is initiallyidentified as a DNA molecule, the sequence of nucleic acids is writtenas a DNA sequence. It is understood that a sequence of nucleotidespresented in text form as a DNA sequence inherently provides descriptionof the corresponding RNA sequence, wherein thymines (T's) within the DNAsequence are replaced with uridines (U's) to get the corresponding RNAsequence of nucleotides. Similarly, it is understood that a sequencepresented in text form as a RNA sequence inherently provides descriptionof the corresponding DNA sequence, wherein uridines (U's) within the RNAsequence are replaced with thymines (T's) to get the corresponding DNAsequence.

Several GPVI nucleic acid ligands obtain via the SELEX method weresequenced and their sequences aligned. Alignment of the sequences shownin FIG. 4 resulted in the identification of 6 unique sequences enrichedthrough the selection process. An alignment of the 6 sequences, referredto as “A” through “F”, shows the presence of a UAA sequence. Moreover,this fully conserved sequence is contained within the sequence:(G/A)UAA. The (G/A)UAA sequence, which is flanked on each side by a GC,giving rise to a conserved GC(G/A)UAAGC sequence.

Secondary structure prediction analysis was then performed for theunique GPVI ligands. Secondary structure contributes to the functionalnature of the ligand. As is well understood by the skilled artisan, thesecondary structure can be described in terms of stem and loopstructures as they occur in the molecule in a 5′ to 3′ direction. Basedon secondary structure prediction as described in Example 2 below,ligands to the GPVI ECD possess a secondary structure that includesthree stems and four loops. The 5′ to 3′ configuration includes a firststem (Stem 1 or S1), a first loop (Loop 1 or L1), a second stem (Stem 2or S2) connecting to the second and a third loop (Loop 3 or L3), thethird loop, a third stem (Stem 3 or S3) and a fourth loop (Loop 4 or L4)(see FIGS. 8A-8C). In some embodiments, the second stem links the first,second and third loops and the third stem links the third and fourthloop. In one embodiment, the third stem is adjacent to the first stem ina 5′-3′ direction. The sequence GC(G/A)UAAGC forms Stem 3 (a GC basepair) and Loop 4 ((G/A)UAA). The sequence GAC forms Loop 1.

Mutational analysis of GPVI ligands identified by SELEX shows that aLoop 3 sequence of UA, UU or UG supports high affinity binding of theligands to GPVI. Accordingly, in some embodiments, the Loop 3 of theGPVI nucleic acid ligand comprises the sequence 5′-YD-3′, wherein Yrepresents a pyrimidine and D represents U, G or A.

In some embodiments, Loop 2 of the GPVI nucleic acid ligand may besubstituted with a spacer using methods known to skilled artisans. Thespacer can be a non-nucleotide spacer which provides a structureanalogous to Loop 2 such that the GPVI ligand maintains its structureand function when Loop 2 is substituted with the spacer. Substitution ofLoop 2 with a hexaethylene glycol spacer provided by incorporation of(9-O-Dimethoxytrityl-triethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (see FIGS.13A-13B) into the GPVI nucleic acid ligand resulted in no loss ofaffinity for GPVI. Accordingly, one having ordinary skill in the artwould understand that Loop 2 can be replaced with a variety ofnon-nucleotide spacers that are commercially available. Examples of suchspacers include, but are not limited to those provided by incorporationof,5′-O-Dimethoxytrityl-1′2′Dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;18-O-Dimethoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite;and12-(4,4′-Dimethoxytrityloxy)dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditeinto the GPVI nucleic acid ligand.

Efficacy of a GPVI ligand in regulating GPVI function or treatingplatelet-mediated disease depends largely upon the ability of the ligandto bind with sufficient affinity to the GPVI protein. Accordingly, afterobtaining GPVI ligands through the SELEX process, each ligand issequenced, and then may be characterized in terms of binding to thetarget molecule. The binding affinity of the ligands herein with respectto the target (GPVI) can be defined in terms of K_(d). The value of thisdissociation constant can be determined directly by well-known methods,such as by radioligand binding methods described in Example 1. It hasbeen observed, however, that for some small oligonucleotides, directdetermination of K_(d) is sometimes difficult, and can lead tomisleadingly high results. Under these circumstances, a competitivebinding assay for the target molecule or other candidate substance canbe conducted with respect to substances known to bind the target orcandidate. The value of the concentration at which 50% inhibition occurs(K_(i)) is, under ideal conditions, equivalent to K_(d). However, in noevent will a K_(i) be less than K_(d). Thus, determination of K_(i), inthe alternative, sets a maximal value for the value of K_(d). Underthose circumstances where technical difficulties preclude accuratemeasurement of K_(d), measurement of K_(i), can conveniently besubstituted to provide an upper limit for K_(d). A K_(i) value can alsobe used to confirm that an ligand of the present binds a target. Incharacterizing GPVI ligand binding properties, specificity may beanalyzed using competition binding or functional assays with known GPVIbinding molecules such as collagen, CRP (collagen related peptide), orconvulxin (Cvx).

In some embodiments, the K_(d) of binding of the ligand to GPVI canrange from between about 1 nM to about 100 nM, from about 10 nM to about50 nM or from about 20 nM to about 0.1 nM. In other embodiments, theK_(d) of binding of a ligand to GPVI is at least 2-fold, 3-fold, 4-fold,5-fold or 10-fold less than the K_(d) of binding of the ligand to anunrelated protein or other accompanying material in the environment. Theunrelated protein could also be a protein having motifs related to thosepresent in GPVI, such as another Ig superfamily member or anotherprotein including a collagen-binding domain or another plateletactivation or adhesion receptor.

As will be discussed in greater detail below, the binding activity ofthe ligand obtained and identified by the SELEX method can be furthermodified or enhanced using a variety of engineering methods.

In some embodiments, the ligand interacts with the extracellular domainof GPVI. The ligand can interfere with collagen binding of the GPVIreceptor. In certain embodiments, the ligand can inhibit intracellularsignaling via the GPVI receptor, including reducing the generation ofinositol trisphosphate or fluctuations in intracellular calcium levels.The ligand can also stabilize or disrupt a conformation of the receptor,such as a dimeric conformation, so that the receptor has a reducedcapacity to interact with collagen or FcRγ. The ligand can affectplatelet activation by collagen or other GPVI agonists. The ligand canalso affect platelet adhesion to collagen or collagen-related peptides.The ligand can affect platelet aggregation induced by collagen or otherGPVI agonists.

The nucleic acid ligands described herein can function as activelyreversible agents. These are agents or pharmaceutically active moleculesthat, after administration to a patient, can be directly controlled bythe administration of a second agent. As described in more detail below,the second agent, referred to herein as a modulator, can shut off orfine-tune the pharmacologic activity of the ligand. As a result, thepharmacologic activity of the ligand can be reversed by means otherthan, for example, drug clearance.

E. Modulators

In some embodiments, the nucleic acid ligands to GPVI are reversible. Inone aspect, the invention provides a method of modulating the activityof a nucleic acid ligand to GPVI by administering a modulator of theGPVI ligand to a host who has been administered the nucleic acid ligand.

Modulators of the present invention include any pharmaceuticallyacceptable agent that can bind to a nucleic acid ligand and modify theinteraction between that ligand and its target molecule (e.g., bymodifying the structure of the nucleic acid ligand) in a desired manner,or which degrades, metabolizes, cleaves, or otherwise chemically altersthe nucleic acid ligand to modify its biological effect. Examples ofmodulators of the present invention include: oligonucleotides, oranalogues thereof, that are complementary to at least a portion of thenucleic acid ligand sequence (including ribozymes or DNAzymes). Otherexamples include peptide nucleic acids (PNA), mopholino nucleic acids(MNA), or locked nucleic acids (LNA); nucleic acid binding proteins orpeptides; oligosaccharides; small molecules; or nucleic acid bindingpolymers, lipids, nanoparticle, or microsphere-based modulators.

Modulators can be designed so as to bind a particular nucleic acidligand with a high degree of specificity and a desired degree ofaffinity. Modulators can also be designed so that, upon binding, thestructure of the ligand is modified to either a more or less activeform. For example, the modulator can be designed such that upon bindingto the targeted nucleic acid ligand, the secondary and/or tertiarystructure of that ligand is altered whereby the ligand can no longerbind to its target molecule or binds to its target molecule with lessaffinity. Alternatively, the modulator can be designed so that, uponbinding, the three dimensional structure of the ligand is altered sothat the affinity of the ligand for its target molecule is enhanced.That is, the modulator can be designed so that, upon binding, astructural motif is modified such that affinity of the ligand isincreased. In another embodiment, a ligand/modulator pair is designedsuch that binding of the modulator to a nucleic acid ligand moleculewhich cannot bind to the target of interest can result in production ofa structural motif within the ligand which thereby allows the ligand tobind to its target molecule.

Modulators can also be designed to nonspecifically bind to a particularnucleic acid ligand or set of nucleic acid ligands with sufficientaffinity to form a complex. Such modulators can generally associate withnucleic acids via charge-charge interactions. Such modulators can alsosimultaneously bind more than one nucleic acid ligand. The modulator canbe designed so that, upon binding to one or more nucleic acid ligands,the structure of the nucleic acid ligand is not significantly changedfrom its active form, but rather, the modulator masks or stericallyprevents association of the nucleic acid ligand with its targetmolecule.

Nucleotide modulators can be of any length that allows effective bindingto the ligand molecule. For example, oligonucleotide modulations canrange in length from about 10 nucleotides (nt) to about 30 nt, fromabout 10 nt to about 20 nt, or from about 15 nt. The nucleotidemodulators may be 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt,16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 n25,26 nt, 27 nt, 28 nt, 29 nt or 30 nt in length. One having ordinary skillin the art can also envision nucleotide modulators having lengthsgreater than 30 nt.

A nucleic acid ligand as described herein possesses an active tertiarystructure, which can be affected by formation of the appropriate stablesecondary structure. Therefore, while the mechanism of formation of aduplex between a complementary oligonucleotide modulator of theinvention and a nucleic acid ligand is similar to formation of a duplexbetween two short linear oligoribonucleotides, both the rules fordesigning such interactions and the kinetics of formation of such aproduct can be impacted by the intramolecular ligand structure.

The rate of nucleation of initial basepair formation between the nucleicacid ligand and oligonucleotide modulator plays a significant role inthe formation of the final stable duplex, and the rate of this step isgreatly enhanced by targeting the oligonucleotide modulator tosingle-stranded loops and/or single-stranded 3′ or 5′ tails present inthe nucleic acid ligand. For the optimal formation of the intermolecularduplex to occur, the free energy is ideally favorable to the formationof the intermolecular duplex with respect to formation of the existingintramolecular duplexes within the targeted nucleic acid ligand.

The modulators described herein of the invention are generallyoligonucleotides which comprise a sequence complementary to at least aportion of the targeted nucleic acid ligand sequence. For example, themodulator oligonucleotide can comprise a sequence complementary to about6 nt to 25 nt, 8 nt to 20 nt, or 10 nt to 15 nt of the targeted ligand.The length of the modulator oligonucleotide can be readily optimizedusing techniques described herein and known to persons having ordinaryskill in the art, taking into account the targeted ligand and the effectsought. The oligonucleotide can be made with nucleotides bearing D or Lstereochemistry, or a mixture thereof. Naturally occurring nucleosidesare in the D configuration.

While the oligonucleotide modulators of the invention include a sequencecomplementary to at least a portion of a nucleic acid ligand, absolutecomplementarity is not required. A sequence “complementary to at least aportion of an nucleic acid ligand,” referred to herein, is a sequencehaving sufficient complementarity to be able to hybridize with thenucleic acid ligand. The ability to hybridize can depend on both thedegree of complementarity and the length of the nucleic acid. Generally,the larger the hybridizing oligonucleotide, the more base mismatcheswith a target ligand it can contain and still form a stable duplex (ortriplex as the case may, be). One skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex. The oligonucleotides of theinvention can be single-stranded DNA or RNA or chimeric mixtures orderivatives or modified versions thereof.

The modulators can include modifications in both the nucleic acidbackbone and structure of individual nucleic acids. In certainembodiments, the modulator is a nucleic acid complementary to at leastone loop region in the ligand. In some embodiments, the modulator is anoligonucleotide having at least a sequence that hybridizes atphysiological conditions to the fourth loop in the two dimensionalstructure, and especially an oligonucleotide including a sequence3′-AUU-5′. In other embodiments, the modulator is an oligonucleotidethat hybridizes under physiological conditions to a first loop in thesecondary structure of the ligand, and in particular an oligonucleotideincluding at least a sequence 3′-CUG-5′. Depending on the desiredfunction of the modulator, the modulator can be designed to disrupt orstabilize the secondary and/or tertiary structure of the nucleic acidligand.

In some embodiments, the modulator is designed to bind to a “suicideposition” on the ligand and thereby disrupt the sequence of the ligand.A suicide position is a single stranded portion of the ligandsusceptible to enzymatic cleavage. In one exemplary embodiment, thesuicide position becomes single stranded and labile upon binding of themodulator to the ligand and can enhance cleavage of the ligand byenzymes in the circulation, such as blood or liver endonucleases. Incertain embodiments, the modulator binds to the ligand after which theligand can no longer interact with its target.

In one exemplary embodiment, the modulator includes a nucleic acidsequence selected from SEQ ID NO:74-88, inclusive.

In some embodiments, a modulator sequence comprises at least onemodified nucleotide. For example, a 2′-O-methyl and 2′-fluoromodification, which can include 2′-O-methyl cytosine, 2′-O-methyluridine, 2′-O-methyl adenosine, 2′-O-methyl guanosine, 2′ fluorocytidine, or 2′ fluoro uridine.

Various strategies can be used to determine the optimal site within anucleic acid ligand for binding by an oligonucleotide modulator. Anempirical strategy can be used in which complimentary oligonucleotidesare “walked” around the nucleic acid ligand. In accordance with thisapproach, oligonucleotides (e.g., 2′-O-methyl or 2′-fluorooligonucleotides) about 15 nucleotides in length can be used that arestaggered by about 5 nucleotides on the ligand (e.g., oligonucleotidescomplementary to 1-15, 6-20, 11-25, etc. of ligand). An empiricalstrategy can be particularly effective because the impact of thetertiary structure of the nucleic acid ligand on the efficiency ofhybridization can be difficult to predict.

Assays described in the Examples that follow can be used to assess theability of the different oligonucleotides to hybridize to a specificnucleic acid ligand, with particular emphasis on the molar excess of theoligonucleotide required to achieve complete binding of the nucleic acidligand. The ability of the different oligonucleotide modulators toincrease the rate of dissociation of the nucleic acid ligand from, orassociation of the ligand with, its target molecule can also bedetermined by conducting standard kinetic studies using, for example,BIACORE assays. Oligonucleotide modulators can be selected such that a5-50 fold molar excess of oligonucleotide, or less, is required tomodify the interaction between the ligand and its target molecule in thedesired manner.

Alternatively, the targeted nucleic acid ligand can be modified so as toinclude a single-stranded tail (3′ or 5′) in order to promoteassociation with an oligonucleotide modulator. Suitable tails cancomprise 1 to 20 nucleotides, 1 to 10 nucleotides, 1 to 5 nucleotides or3 to 5 nucleotides. Tails may also be modified (e.g., a 2′-O-methyl and2′-fluoro modification, which can include 2′-O-methyl cytosine,2′-O-methyl uridine, 2′-O-methyl adenosine, 2′-O-methyl guanosine, 2′fluoro cytidine, or 2′ fluoro uridine). Tailed ligands can be tested inbinding and bioassays (e.g., as described in the Examples that follow)to verify that addition of the single-stranded tail does not disrupt theactive structure of the nucleic acid ligand. A series ofoligonucleotides (for example, 2′-O-methyl oligonucleotides) that canform, for example, 1, 2, 3, 4 or 5 base pairs with the tail sequence canbe designed and tested for their ability to associate with the tailedligand alone, as well as their ability to increase the rate ofdissociation of the ligand from, or association of the ligand with, itstarget molecule. Scrambled sequence controls can be employed to verifythat the effects are due to duplex formation and not non-specificeffects.

In another embodiment, the modulator is a ribozyme or a DNAzyme.Enzymatic nucleic acids act by first binding to a target RNA or DNA.Such binding occurs through the target binding portion of an enzymaticnucleic acid which is held in close proximity to an enzymatic portion ofa molecule that acts to cleave the target RNA. Thus, the enzymaticnucleic acid first recognizes and then binds a target RNA or DNA throughcomplementary base-pairing, and once bound to the correct site, actsenzymatically to cut the target RNA, thereby allowing for inactivationof RNA ligands. There are at least five classes of ribozymes that eachdisplay a different type of specificity. For example, Group I Intronsare about 300 to >1000 nucleotides in size and require a U in the targetsequence immediately 5′ of the cleavage site and binds 4-6 nucleotidesat the 5′-side of the cleavage site. Another class is RNaseP RNA (M1RNA), which are about 290 to 400 nucleotides in size. A third class isHammerhead Ribozymes, which are about 30 to 40 nucleotides in size. Theyrequire the target sequence UH (where H is not G) immediately 5′ of thecleavage site and bind a variable number of nucleotides on both sides ofthe cleavage site. A fourth class is the Hairpin Ribozymes, which areabout 50 nucleotides in size. They require the target sequence GUCimmediately 3′ of the cleavage site and bind 4 nucleotides at the5′-side of the cleavage site and a variable number to the 3′-side of thecleavage site. A fifth group is Hepatitis Delta Virus (HDV) Ribozymes,which are about 60 nucleotides in size. DNAzymes are single-stranded,and cleave both RNA and DNA. A general model for the DNAzyme has beenproposed, and is known as the “10-23” model. DNAzymes following the“10-23” model have a catalytic domain of 15 deoxyribonucleotides,flanked by two substrate-recognition domains of seven to ninedeoxyribonucleotides each.

In another embodiment, the modulator itself is a nucleic acid ligand. Inthis embodiment, a first ligand is generated that binds to the desiredtherapeutic target. In a second step, a second ligand that binds to thefirst ligand is generated using the SELEX process described herein oranother process, and modulates the interaction between the therapeuticligand and the target. In one embodiment, the second ligand deactivatesthe effect of the first ligand.

In another exemplary embodiment, the modulator is a PNA, MNA, LNA, orPCO based modulator. Nucleobases of the oligonucleotide modulators ofthe invention can be connected via internucleobase linkages, e.g.,peptidyl linkages (as in the case of peptide nucleic acids (PNAs);Nielsen et al. (1991) Science 254, 1497 and U.S. Pat. No. 5,539,082) andmorpholino linkages (Qin et al., Antisense Nucleic Acid Drug Dev. 10, 11(2000); Summerton, Antisense Nucleic Acid Drug Dev. 7, 187 (1997);Summerton et al., Antisense Nucleic Acid Drug Dev. 7, 63 (1997); Tayloret al., J Biol. Chem. 271, 17445 (1996); Partridge et al., AntisenseNucleic Acid Drug Dev. 6, 169 (1996)), or by any other natural ormodified linkage. The oligonucleobases can also be Locked Nucleic Acids(LNAs). Nielsen et al., J Biomol Struct Dyn 17, 175 (1999); Petersen etal., J Mol Recognit 13, 44 (2000); Nielsen et al., Bioconjug Chem 11,228 (2000).

PNAs are compounds that are analogous to oligonucleotides, but differ incomposition. In PNAs, the deoxyribose backbone of oligonucleotide isreplaced with a peptide backbone. Each subunit of the peptide backboneis attached to a naturally-occurring or non-naturally-occurringnucleobase. PNA often has an achiral polyamide backbone consisting ofN-(2-aminoethyl)glycine units. The purine or pyrimidine bases are linkedto each unit via a methylene carbonyl linker (1-3) to target thecomplementary nucleic acid. PNA binds to complementary RNA or DNA in aparallel or antiparallel orientation following the Watson-Crickbase-pairing rules. The uncharged nature of the PNA oligomers enhancesthe stability of the hybrid PNA/DNA (RNA) duplexes as compared to thenatural homoduplexes.

Morpholino nucleic acids are so named because they are assembled frommorpholino subunits, each of which contains one of the four geneticbases (adenine, cytosine, guanine, and thymine) linked to a 6-memberedmorpholine ring. Eighteen to twenty-five subunits of these four subunittypes are joined in a specific order by non-ionic phosphorodiamidateintersubunit linkages to give a morpholino oligo.

LNA is a class of DNA analogues that possess some features that make ita prime candidate for modulators of the invention. The LNA monomers arebi-cyclic compounds structurally similar to RNA-monomers. LNA share mostof the chemical properties of DNA and RNA, it is water-soluble, can beseparated by gel electrophoreses, ethanol precipitated etc (Tetrahedron,54, 3607-3630 (1998)). However, introduction of LNA monomers into eitherDNA or RNA oligos results in high thermal stability of duplexes withcomplementary DNA or RNA, while, at the same time obeying theWatson-Crick base-pairing rules.

Pseudo-cyclic oligonucleobases (PCOs) can also be used as a modulator inthe present invention (see U.S. Pat. No. 6,383,752). PCOs contain twooligonucleotide segments attached through their 3′-3′ or 5′-5′ ends. Oneof the segments (the “functional segment”) of the PCO has somefunctionality (e.g., complementarity to a target RNA). Another segment(the “protective segment”) is complementary to the 3′- or 5′-terminalend of the functional segment (depending on the end through which it isattached to the functional segment). As a result of complementaritybetween the functional and protective segment segments, PCOs formintramolecular pseudo-cyclic structures in the absence of the targetnucleic acids (e.g., RNA). PCOs are more stable than conventionaloligonucleotides because of the presence of 3′-3′ or 5′-5′ linkages andthe formation of intramolecular pseudo-cyclic structures.Pharmacokinetic, tissue distribution, and stability studies in micesuggest that PCOs have higher in vivo stability than and,pharmacokinetic and tissue distribution profiles similar to, those ofPS-oligonucleotides in general, but rapid elimination from selectedtissues. When a fluorophore and quencher molecules are appropriatelylinked to the PCOs of the present invention, the molecule will fluorescewhen it is in the linear configuration, but the fluorescence is quenchedin the cyclic conformation. This feature can be used to screen PCO's aspotential modulators.

In another exemplary embodiment, the modulators are peptide-basedmodulators. Peptide-based modulators of nucleic acid ligands representan alternative molecular class of modulators to oligonucleotides ortheir analogues. This class of modulators are particularly useful ifsufficiently active oligonucleotide modulators of a target nucleic acidligand cannot be isolated due to the lack of sufficient single-strandedregions to promote nucleation between the target and the oligonucleotidemodulator. In addition, peptide modulators provide differentbioavailabilities and pharmacokinetics than oligonucleotide modulators.In one exemplary embodiment the modulator is a protamine (Oney et al.,2009, Nat. Med. 15:1224-1228). Protamines are soluble in water, are notcoagulated by heat, and comprise arginine, alanine and serine (most alsocontain proline and valine and many contain glycine and isoleucine).Modulators also include protamine variants (see e.g., Wakefield et al,J. Surg. Res. 63:280 (1996)) and modified forms of protamine, includingthose described in U.S. Publication No. 20040121443. Other modulatorsinclude protamine fragments, such as those described in U.S. Pat. No.6,624,141 and U.S. Publication No. 20050101532. Modulators also include,generally, peptides that modulate the activity of heparin, otherglycosaminoglycans or proteoglycans (see, for example, U.S. Pat. No.5,919,761). In one exemplary embodiment, modulators are peptides thatcontain cationic-NH groups permitting stabilizing charge-chargeinteractions such as poly-L-lysine and poly-L-ornithine.

Several strategies to isolate peptides capable of binding to and therebymodulating the activity of a target nucleic acid ligand are available.For example, encoded peptide combinatorial libraries immobilized onbeads have been described, and have been demonstrated to containpeptides able to bind viral RNA sequences and disrupt the interactionbetween the viral RNA and a viral regulatory protein that specificallybinds said RNA (Hwang et al. Proc. Natl. Acad. Sci. USA, 1999,96:12997). Using such libraries, modulators of nucleic acid ligands canbe isolated by appending a label to the target nucleic acid ligand andincubating together the labeled-target and bead-immobilized peptidelibrary under conditions in which binding between some members of thelibrary and the nucleic acid are favored. The binding of the nucleicacid ligand to the specific peptide on a given bead causes the bead tobe “colored” by the label on the nucleic acid ligand, and thus enablethe identification of peptides able to bind the target by simpleisolation of the bead. The direct interaction between peptides isolatedby such screening methods and the target nucleic acid ligand can beconfirmed and quantified using any number of the binding assaysdescribed to identify modulators of nucleic acid ligands. The ability ofsaid peptides to modulate the activity of the target nucleic acid ligandcan be confirmed by appropriate bioassays.

In an additional embodiment, the modulators are oligosaccharide basedmodulators. Oligosaccharides can interact with nucleic acids. Forexample, the antibiotic aminoglycosides are products of Streptomycesspecies and interact specifically with a diverse array of RNA moleculessuch as various ribozymes, RNA components of ribosomes, and HIV-1's TARand RRE sequences. Thus oligosaccharides can bind to nucleic acids andcan be used to modulate the activity of nucleic acid ligands.

In another embodiment, the modulator is a small molecule basedmodulator. A small molecule that intercalates between the ligand and thetarget or otherwise disrupts or modifies the binding between the ligandand target can also be used as the therapeutic regulator. Such smallmolecules can be identified by screening candidates in an assay thatmeasures binding changes between the ligand and the target with andwithout the small molecule, or by using an in vivo or in vitro assaythat measures the difference in biological effect of the ligand for thetarget with and without the small molecule. Once a small molecule isidentified that exhibits the desired effect, techniques such ascombinatorial approaches can be used to optimize the chemical structurefor the desired regulatory effect.

In a further exemplary embodiment, the modulator is a nucleic acidbinding polymer, lipid, nanoparticle or microsphere. In furthernon-limiting examples, the modulator can be selected from the groupconsisting of: 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC);dilauroylethylphosphatidylcholine (EDLPC); EDLPC/EDOPC; pyridiniumsurfactants; dioleoylphosphatidyl-ethanolamine (DOPE);(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide (GAP-DLRIE) plus the neutral co-lipiddioleoylphosphatidylethanolamine (DOPE) (GAP-DLRIE/DOPE);(±)-N,N-dimethyl-N-[2-(sperminecarboxamido)ethyl]-2,3-bis(diocyloxy-1-propaniminium petahydrochloride(DOSPA); dilauroylethylphosphatidylcholine (EDLPC); Ethyldimyristoylphosphatidylcholine (EDMPC);(±)-N,N,N-trimethyl-2,3-bis(z-octadec-9-ene-oyloxy)-1-propanaminiumchloride (DOTAP);(±)-N-2-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminiumbromide (DMRIE);(±)-N,N,N-trimethyl-2,3-bis(z-octadec-9-enyloxy)-1-propanaminiumchloride (DOTMA); 5-carboxyspermylglycine dioctadecyl-amide (DOGS);dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES); 1,3dioleoyloxy-2-(6-carboxyspermyl)-propyl-amid (DOSPER);tetramethyltetrapalmitoyl spermine (TMTPS); (tetramethyltetraoleylspermine (TMTOS); tetramethlytetralauryl spermine (TMTLS);tetramethyltetramyristyl spermine (TMTMS); tetramethyldioleyl spermine(TMDOS); diphytanoylphosphatidyl-ethanolamine (DPhPE); and(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide (GAP-DLRIE).

In other embodiments, the modulator is selected from the groupconsisting of: chitosan; a chitosan derivative;1,5-dimethyl-1,5-diazaundecamethylene polymethobromide;polyoxyethylene/polyoxypropylene block copolymers; poly-L-lysine;polyamidoamine (PAMAM); β-cyclodextrin-containing polycation (CDP);β-cyclodextrin-containing polycation (imidazole-containing variant)(CDP-Im); polyphosphoramidate polymer (8 kDa, 30 kDa) (PPA-DPA 8k,PPA-DPA 30k); polybrene; spermine; PEG-block-PLL-dendrimers;polyethylenimine (PEI); mannose-PEI; transferin-PEI; linera-PEI (lPEI);gelatin; methacrylate/methacrylamide; poly(beta-amino esters);polyelectrolyte complexes (PEC); poly(vinalyamine) (PVA); Collagen;polypropylene imine (PPI); polyallylamine; polyvinylpyridine;aminoacetalized poly(vinyl alcohol); acrylic or methacrylic polymer;Newkome dendrimer; polyphenylene; dimethyldioctadecylammonium bromide(DAB); cetyltrimethylammonium bromide (CTAB); albumin; acid-treatedgelatin; polylysine; polyornithine; polyarginine; DEAE-cellulose;DEAE-dextran; and poly(N,N-dimethylaminoethylmethacrylate); andpolypropylamine (POPAM).

In one embodiment, the modulator is selected from chitosan and chitasanderivatives. Chitosan derivatives include water soluble chitosannanoparticles (such as described in U.S. Pat. No. 6,475,995; US PatentApplication No. 2006/0013885; Limpeanchob et al, (2006) Efficacy andToxicity of Amphotericin B-Chitosan Nanoparticles; Nareusan UniversityJournal 14(2):27-34). Given the polycationic nature of the chitosanpolymer (essentially a very large polyamine polymer composed ofrepeating glucosamine monomers), chitosan may be used to aggregateand/or encapsulate ligands into a polyelectrolyte complex in vivofollowing injection into a host. This is based in part on interactionsof the primary amines found on chitosan and the phosphodiester backboneof the ligand.

In certain embodiments, the primary amines on the chitosan polymer canbe substantially modified to alter the water solubility and chargestate. Chitosan derivatives include trimethyl chitosan chloride (TMC),which can be synthesized at different degrees of quaternization;mono-carboxymethylated chitosan (MCC) which is a polyampholytic polymer;glutaraldehyde cross-linked derivative (CSGA); thiolated chitosan (Lee,et al. (2007) Pharm. Res. 24:157-67); glycol chitosan (GC), a chitosanderivative conjugated with ethylene glycol (Lee, et al. (2007) Int J.Pharm.); [N-(2-carboxybenzyl)chitosan (CBCS) (Lin, et al. (2007)Carbohydr Res. 342(1):87-95); a beta-cyclodextrin-chitosan polymer(Venter, et al. (2006) Int J Pharm. 313(1-2):36-42);O-carboxymethylchitosan; N,O-carboxymethyl chitosan; or a chitosanchemically modified by introducing xanthate group onto its backbone.

In one embodiment, empty chitosan nanoparticles are generated and usedas modulators. Chitosan or chitosan derivatives of molecular weightrange of 10,000 Da to >1,000,000 Da may be used. In certain embodiments,the chitosan is of 500,000 Da or less. In certain embodiments, thechitosan is of 100,000 Da or less. In some embodiments, the compound isbetween 10,000 and 100,000 Da, between 10,000 and 90,000, between 10,000and 80,000, between 20,000 and 70,0000, between 30,000 and 70,000, about30,000, about 40,000, about 50,000 or about 60,000 Da.

In some embodiments, chitosan polymers containing different degrees ofdeacetylated primary amines are used. In these embodiments, thedifferent degrees of deacetylation alters the charge state of thepolymer and thereby the binding properties of the polymer. Upon contactof the chitosan nanoparticle with ligands in the host, ligands may bindwith and become trapped on the nanoparticle surface, or enter thenanoparticle and become encapsulated by ionic interactions.

In another embodiment, the modulator is a polyphosphate polymermicrosphere. In certain embodiments, the modulator is a derivative ofsuch a microsphere such as poly(L-lactide-co-ethyl-phosphite) orP(LAEG-EOP) and others, as described in U.S. Pat. No. 6,548,302. Suchpolymers can be produced to contain a variety of functional groups aspart of the polymeric backbone. In one example, the polymers may containquaternary amines with a positive charge at physiologic pH, such thatthey can complex or encapsulate one or more nucleic acids upon contact.In certain embodiments, the polymers do not contain positive charges.

The present invention also provides methods to identify the modulatorsof nucleic acid GPVI ligands. Modulators can be identified in general,through binding assays, molecular modeling, or in vivo or in vitroassays that measure the modification of biological function. In oneembodiment, the binding of a modulator to a nucleic acid is determinedby a gel shift assay. In another embodiment, the binding of a modulatorto a nucleic acid ligand is determined by a BIACORE assay.

Standard binding assays can be used to identify and select modulators ofthe invention. Non-limiting examples are gel shift assays and BIACOREassays. That is, test modulators can be contacted with the nucleic acidligands to be targeted under test conditions or typical physiologicalconditions and a determination made as to whether the test modulator infact binds the ligand. Test modulators that are found to bind thenucleic acid ligand can then be analyzed in an appropriate bioassay(which will vary depending on the ligand and its target molecule, forexample coagulation tests) to determine if the test modulator can affectthe biological effect caused by the ligand on its target molecule.

The Gel-Shift assay is a well-known technique used to assess bindingcapability. For example, a DNA fragment containing the test sequence isfirst incubated with the test protein or a mixture containing putativebinding proteins, and then separated on a gel by electrophoresis. If theDNA fragment is bound by protein, it will be larger in size and itsmigration will therefore be retarded relative to that of the freefragment. For example, one method for a electrophoretic gel mobilityshift assay can be (a) contacting in a mixture a nucleic acid bindingprotein with a non-radioactive or radioactive labeled nucleic acidmolecule comprising a molecular probe under suitable conditions topromote specific binding interactions between the protein and thenucleic acid in forming a complex, wherein said probe is selected fromthe group consisting of dsDNA, ssDNA, and RNA; (b) electrophoresing themixture; and (c) detecting the complex bound to the membrane bydetecting the non-radioactive or radioactive label in the complex.

The BIACORE technology measures binding events on the sensor chipsurface, so that the interactant attached to the surface determines thespecificity of the analysis. Testing the specificity of an interactioninvolves simply analyzing whether different molecules can bind to theimmobilized interactant. Binding gives an immediate change in thesurface plasmon resonance (SPR) signal, so that it is directly apparentwhether an interaction takes place or not. SPR-based biosensors monitorinteractions by measuring the mass concentration of biomolecules closeto a surface. The surface is made specific by attaching one of theinteracting partners. Sample containing the other partner(s) flows overthe surface: when molecules from the sample bind to the interactantattached to the surface, the local concentration changes and an SPRresponse is measured. The response is directly proportional to the massof molecules that bind to the surface.

SPR arises when light is reflected under certain conditions from aconducting film at the interface between two media of differentrefractive index. In the BIACORE technology, the media are the sampleand the glass of the sensor chip, and the conducting film is a thinlayer of gold on the chip surface. SPR causes a reduction in theintensity of reflected light at a specific angle of reflection. Thisangle varies with the refractive index close to the surface on the sideopposite from the reflected light. When molecules in the sample bind tothe sensor surface, the concentration and therefore the refractive indexat the surface changes and an SPR response is detected. Plotting theresponse against time during the course of an interaction provides aquantitative measure of the progress of the interaction. The BIACOREtechnology measures the angle of minimum reflected light intensity. Thelight is not absorbed by the sample: instead the light energy isdissipated through SPR in the gold film. SPR response values areexpressed in resonance units (RU). One RU represents a change of 0.0001°in the angle of the intensity minimum, for most proteins, this isroughly equivalent to a change in concentration of about 1 pg/mm² on thesensor surface. The exact conversion factor between RU and surfaceconcentration depends on properties of the sensor surface and the natureof the molecule responsible for the concentration change.

There are a number of other assays that can determine whether anoligonucleotide or analogue thereof, peptide, polypeptide,oligosaccharide or small molecule can bind to the ligand in a mannersuch that the interaction with the target is modified. For example,electrophoretic mobility shift assays (EMSAs), titration calorimetry,scintillation proximity assays, sedimentation equilibrium assays usinganalytical ultracentrifugation (see for eg.www.cores.utah.edu/interaction), fluorescence polarization assays,fluorescence anisotropy assays, fluorescence intensity assays,fluorescence resonance energy transfer (FRET) assays, nitrocellulosefilter binding assays, ELISAs, ELONAs (see, for example, U.S. Pat. No.5,789,163), RIAs, or equilibrium dialysis assays can be used to evaluatethe ability of an agent to bind to a nucleic acid ligand. Direct assaysin which the interaction between the agent and the nucleic acid ligandis directly determined can be performed, or competition or displacementassays in which the ability of the agent to displace the ligand from itstarget can be performed (for example, see Green, Bell and Janjic,Biotechniques 30(5), 2001, p 1094 and U.S. Pat. No. 6,306,598). Once acandidate modulating agent is identified, its ability to modulate theactivity of a nucleic acid ligand for its target can be confirmed in abioassay. Alternatively, if an agent is identified that can modulate theinteraction of a ligand with its target, such binding assays can be usedto verify that the agent is interacting directly with the ligand and canmeasure the affinity of said interaction.

In another embodiment, mass spectrometry can be used for theidentification of a modulator that binds to a nucleic acid ligand, thesite(s) of interaction between the modulator and the nucleic acidligand, and the relative binding affinity of agents for the ligand (seefor example U.S. Pat. No. 6,329,146). Such mass spectral methods canalso be used for screening chemical mixtures or libraries, especiallycombinatorial libraries, for individual compounds that bind to aselected target ligand that can be used in as modulators of the ligand.Furthermore, mass spectral techniques can be used to screen multipletarget nucleic acid ligands simultaneously against, e.g. a combinatoriallibrary of compounds. Moreover, mass spectral techniques can be used toidentify interaction between a plurality of molecular species,especially “small” molecules and a molecular interaction site on atarget ligand.

In vivo or in vitro assays that evaluate the effectiveness of amodulator in modifying the interaction between a nucleic acid ligand anda target are specific for the disorder being treated. There are amplestandard assays for biological properties that are well known and can beused. Examples of biological assays are provided in the patents cited inthis application that describe certain nucleic acid ligands for specificapplications.

In some embodiments, a modulator is a small molecule. For example, incertain embodiments, a nucleic acid ligand is linked to a biotinmolecule. In those instances, a streptavadin or avidin is administeredto bind to and reverse the effects of the ligand (see Savi et. al. JThrombosis and Haemostasis, 6: 1697-1706). Avidin is a tetramericprotein produced in the oviducts of birds, reptiles and amphibians whichis deposited in the whites of their eggs. Streptavidin is a tetramericprotein purified from the bacterium Streptomyces avidinii. Thetetrameric protein contains four identical subunits (homotetramer) eachof which can bind to biotin (Vitamin B₇, vitamin H) with a high degreeof affinity and specificity.

In certain embodiments, a modulator is a cationic molecule. In certainembodiments, the ligand forms a guanine quartet (G-quartet orG-quadruplex) structure. These structures are bound by cationicmolecules. In certain embodiments, the molecules are metal chelatingmolecules. In some embodiments, the modulator is a porphyrin. In someembodiments, the compound is TMPyP4. See Joachimi, et. al. JACS 2007,129, 3036-3037 and Toro, et. al. Analytical Biochemistry 2008, Aug. 1,379 (1) 8-15.

In one embodiment, the modulator has the ability to substantially bindto a nucleic acid ligand in solution at modulator concentrations of lessthan ten (10.0) micromolar (uM), one (1.0) micromolar (uM), preferablyless than 0.1 uM, and more preferably less than 0.01 uM. By“substantially” is meant that at least a 50 percent reduction in targetbiological activity is observed by modulation in the presence of the atarget, and at 50% reduction is referred to herein as an IC₅₀ value.

F. Optimizing Ligands and Modulators

In order for a ligand to be suitable for use as a therapeutic, theligand is preferably inexpensive to synthesize, safe for use in a host,and stable in vivo. Wild-type RNA and DNA oligonucleotides are typicallynot stable in vivo because of their susceptibility to degradation bynucleases. Resistance to nuclease degradation can be greatly increasedby the incorporation of modifying groups at the 2′-position.

2′-fluoro or amino groups may be incorporated into oligonucleotide poolsfrom which ligands have been subsequently selected. In the presentdisclosure, 2′-fluoropyrimidines were used in an in vitro transcriptionreaction to generate an initial oligonucleotide pool for ligandselection (see Example 1). However, resultant ligands selected from suchlibraries containing 2′-hydroxyl sugars at each purine position, sowhile more stable in vivo than a comparable RNA or DNA ligand, requireadditional optimization. Accordingly, the ligands identified using themethods described herein are subsequently modified in a variety of waysto obtain a ligand which has enhanced function and stability, as well asincreased feasibility for large-scale manufacturing processes.

After initial identification of the ligands (via e.g., SELEX) and themodulators (e.g., design based on sequence complementarity), the ligandsand modulators can be modified or engineered to improve their desiredstructure, function and/or stability by a variety of means. Theseinclude, but are not limited to, substituting particular sugar residues,changing the composition and size of particular regions and/orstructures in the ligand, and designing ligands that can be moreeffectively regulated by a modulator.

The design and optimization of a nucleic acid ligand involves anappreciation for the secondary structure of the ligand as well as therelationship between the secondary structure and the modulator control.Unlike conventional methods of modifying nucleic acids, the design ofthe ligands to the GPVI protein may include consideration of the impactof changes to the ligand on the design of potential modulators. If aligand is modified by truncation, for example, the correspondingmodulator should be designed to control the truncated ligand.

The secondary structure of ligands identified through the SELEX processcan be predicted by various methods known to persons having ordinaryskill in the art. For example, each sequence may be analyzed using asoftware program such as Mfold (mfold.bioinfo.rpi.edu; see also Zuker,2003, Nucleic Acids Res. 31:3406-3415 and Mathews, et al., 1999, J. Mol.Biol. 288:911-940). Subsequently, comparative sequence analysis of thevarious selected sequences can be used to align the sequences based uponconserved consensus secondary structural elements to arrive at apredicted secondary consensus structure for GPVI ligands (see Example2). An analysis such as that described above allows one to design andtest variants of the sequences obtained through SELEX to generateligands with enhanced function and stability.

GPVI nucleic acid ligands of the present invention can be modified byvarying overall ligand length as well as the lengths of the stem andloop structures. For example, ligand truncations may be generated inwhich a portion of the 5′ and/or 3′ end of a ligand is deleted from theligand selected in the SELEX process. To determine the extent oftruncations which are tolerated by a ligand, one method used can be toheat anneal an oligonucleotide (e.g. a DNA oligonucleotide)complementary to a 5′ or 3′ terminal region of the ligand, then comparebinding of the ligand with and without the annealed oligonucleotide. Ifno significant binding difference is observed between the ligand withand the ligand without the annealed oligonucleotide, this suggests thatthe annealed portion of the ligand is dispensable for binding of theligand to the target protein. This method can be performed usingoligonucleotides which anneal to various lengths of the 5′ or 3′ ends ofthe ligand to determine 5′ and 3′ boundaries which provide a fullyfunctional ligand.

In another embodiment, the design includes decreasing the size of theligand. In another embodiment, the size of the modulator is changed inrelation to the size of the ligand. In yet another embodiment, guaninestrings are reduced to less than four guanine, or less than threeguanine, or less than two guanine or no guanines. However, the jointeffect of these changes must meet the challenge of creating a ligandthat provides adequate activity but is easily neutralized by themodulator.

For targeting of a modulator, an improved ligand can also be modified soas to include a single-stranded tail (3′ or 5′) in order to promoteassociation with an oligonucleotide modulator. Suitable tails cancomprise 1 nt to 20 nt, preferably, 1 nt to 10 nt, 1 nt to 5 nt or 3 ntto 5 nt. It is readily understood that such tails may included modifiednucleotides as described in more detail below.

Tailed ligands can be tested in binding and bioassays (e.g., asdescribed below) to verify that addition of the single-stranded taildoes not disrupt the active structure of the ligand. A series ofoligonucleotides (for example, 2′-O-methyl oligonucleotides) that canform, for example, 1, 3 or 5 base-pairs with the tail sequence can bedesigned and tested for their ability to associate with the tailedligand alone, as well as their ability to increase the rate ofdissociation of the ligand from, or association of the ligand with, itstarget molecule. Scrambled sequence controls can be employed to verifythat the effects are due to duplex formation and not non-specificeffects.

Determination of a consensus structure also facilitates engineering ofligands to identify one or more nucleotides which may enhance ordecrease ligand structure and function. For example, one may moreefficiently identify and test nucleotide additions, deletions andsubstitutions to specific stem and loop structures (see Example 3).

Knowledge of a consensus secondary structure also allows one to avoidmodifications which may be detrimental to ligand structure and function.For example, certain modifications may be conserved within the consensussecondary structure, such a 2′-fluoro within a stem or loop region. Inthese instances, removal of a 2′-fluoro from the stem or loop of anligand may result in the loss of activity.

In certain embodiments, the ligands are nucleic acid molecules selectedfrom Tables 1-7, including truncates and substantially homologoussequences thereof. As used herein, in the context of homologous regions,a “substantially homologous” sequence is one that forms the samesecondary structure by Watson-Crick base pairing within a particularmolecule. In certain embodiments, sequences are “substantiallyhomologous” if they share at least 80%, 85% or more sequence identity,such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to a specified ligand. In the context of a nucleic acid ligandof a specified length, such as 50 or less nucleotides, a homologoussequence can be found in any region that allows Watson-Crick binding toform the same secondary structure, regardless of sequence identitywithin the specific region.

Ligands may also be designed to have a suicide position, which allowsmore effective regulation by paired modulators. Upon binding of theligand by the modulator, the suicide position becomes single strandedand labile, thereby facilitating cleavage of the ligand by enzymesnaturally present in the blood, such as blood or liver endonucleases.This provides a means for effective and substantially immediateelimination of the active ligand from circulation.

Chemical Modifications

One problem encountered in the therapeutic use of nucleic acids is thatoligonucleotides in their phosphodiester form may be quickly degraded inbody fluids by intracellular and extracellular enzymes such asendonucleases and exonucleases before the desired effect is manifest.Certain chemical modifications of the nucleic acid ligand can increasethe in vivo stability of the nucleic acid ligand or to enhance or tomediate the delivery of the nucleic acid ligand. Additionally, certainchemical modifications can increase the affinity of the nucleic acidligand for its target, by stabilizing or promoting the formation ofrequired structural elements within the nucleic acid ligand or providingadditional molecular interactions with the target molecule.

Modifications of the ligands can include, but are not limited to, thosewhich provide chemical groups that incorporate additional charge,polarizability, hydrophobicity, hydrogen bonding, electrostaticinteractions, and functionality to the nucleic acid ligand bases or tothe ligand as a whole. Such modifications include, but are not limitedto, 2′-position sugar modifications, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atexocyclic amines, substitution of 4-thiouridine, substitution of 5-bromoor 5-iodo-uracil, backbone modifications, phosphorothioate or alkylphosphate modifications, methylations, unusual base-pairing combinationssuch as the isobases isocytidine and isoguanidine and the like.Modifications can also include 3′ and 5′ modifications such as capping.

The SELEX method encompasses the identification of high-affinity nucleicacid ligands containing modified nucleotides conferring improvedcharacteristics on the ligand, such as improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX-identified nucleic acid ligands containingmodified nucleotides are described in U.S. Pat. No. 5,660,985 thatdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the 5- and 2′-positions of pyrimidines. U.S. Pat. No.5,580,737 describes specific nucleic acid ligands containing one or morenucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F), and/or2′-O-methyl (2′-OMe). U.S. Pat. No. 5,756,703, describesoligonucleotides containing various 2′-modified pyrimidines.

The SELEX method encompasses combining selected oligonucleotides withother selected oligonucleotides and non-oligonucleotide functional unitsas described in U.S. Pat. Nos. 5,637,459 and 5,683,867. U.S. Pat. No.5,637,459 describes highly specific nucleic acid ligands containing oneor more nucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F),and/or 2′-O-methyl (2′-OMe). The SELEX method further encompassescombining selected nucleic acid ligands with lipophilic orNon-Immunogenic, High Molecular Weight compounds in a diagnostic ortherapeutic complex as described in U.S. Pat. No. 6,011,020.

Where the nucleic acid ligands are derived by the SELEX method, themodifications can be pre- or post-SELEX modifications. Pre-SELEXmodifications can yield ligands with both specificity for its target andimproved in vivo stability. Post-SELEX modifications made to 2′-hydroxyl(2′-OH) nucleic acid ligands can result in improved in vivo stabilitywithout adversely affecting the binding capacity of the nucleic acidligands. In one embodiment, the modifications of the ligand include a3′-3′ inverted phosphodiester linkage at the 3′ end of the molecule, and2′ fluoro (2′-F), 2′ amino (2′-NH₂), 2′ deoxy, and/or 2′ O methyl(2′-OMe) modification of some or all of the nucleotides.

The ligands described herein were initially generated via SELEX usinglibraries of transcripts in which the C and U residues were 2′-fluorosubstituted and the A and G residues were 2′-OH. While suchmodifications generate ligand molecules suitable for screening, the high2′ hydroxyl content make them unsuitable for drug development candidatesdue to the fact that these positions can be very sensitive to nucleasedegradation in vivo, limiting the maximal concentration that can beachieved post-parenteral administration as well as their circulatinghalf-life. Accordingly, once functional sequences are identified, suchas through the SELEX method, individual residues can be tested fortolerance to substitutions by assessing the effects of thesesubstitutions on ligand structure, function and stability.

In certain embodiments, the nucleic acids making up the ligand includemodified sugars and/or modified bases. In certain embodiments, themodifications include stabilizing modifications such as 2′-stabilizingmodifications. In one embodiment, 2′-stabilizing modifications caninclude 2′-fluoro, 2′ deoxy or 2′-O-methyl modifications on the sugarring.

In one embodiment, the design includes decreasing the 2′-hydroxylcontent of the ligand or the modulator, or both. In another embodiment,the design includes decreasing the 2′-fluoro content of the ligand orthe modulator, or both. In another embodiment, the design includesincreasing the 2′-O-methyl content of the ligand or the modulator, orboth.

The oligonucleotide can comprise at least one modified base moiety whichis selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2α-thiouracil, β-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N &isopentenyladenine, uracil oxyacetic acid, wybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, -uracil-5-oxyacetic acid methylester,uracil oxyacetic acid (v), 5-methyl thiouracil, 3-(3-amino-3-Ncarboxypropyl) and 2,6-diaminopurine.

The oligonucleotides of the presently described ligands and modulatorscan comprise modified sugar groups, for example, one or more of thehydroxyl groups is replaced with halogen, aliphatic groups, orfunctionalized as ethers or amines. In one embodiment, the 2′-positionof the furanose residue is substituted by any of an O-methyl, O-alkyl,O-allyl, S-alkyl, S-allyl, or halo group. In another embodiment, thenucleic acid ligand or modulator of the invention can comprise at leastone modified sugar moiety selected from the group including, but notlimited to, arabinose, 2-fluoroarabinose, xylulose, hexose,2′-fluororibose, 2′-O-methylribose, 2′-O-methoxyethylribose,2′-O-propylribose, 2′-O-methylthioethylribose,2′-O-diethylaminooxyethylribose, 2′-O-(3-aminopropyl)ribose,2′-O-(dimethylaminopropyl)ribose, 2′-O-(methylacetamido)ribose, and2′-O-(dimethylaminoethyloxyethyl)ribose.

The ligand or modulator can comprise at least one modified phosphatebackbone selected from the group including, but not limited to, aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphorodiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

The ligand molecule, which comprises stem and loop structures, may befurther stabilized for therapeutic use by the substitution of one ormore nucleic acid loop structures with a more stable loop structure. Forexample, for the GPVI ligands described herein, it was discovered thatsubstitution of Loop 2 with a hexaethylene glycol spacer resulted inGPVI ligands with similar affinity and anti-GPVI ligands withnucleotidyl loop. FIG. 13A illustrates the starting phosphoramidite fora hexaethylene glycol linker used in synthesis. FIG. 13B illustrates thehexaethylene glycol spacer when incorporated between two nucleotides ofa nucleic acid ligand.

In pharmaceutical compositions the ligands can be provided in forms,such as salt forms that improve solubility or bioavailability.

Any of the oligonucleotides of the invention can be synthesized bystandard methods known in the art, e.g. by use of an automated DNAsynthesizer (such as are commercially available from, for example,Biosearch, Applied Biosystems).

Ligands and modifiers are described herein using abbreviations readilyunderstood by a skilled artisan and noted as follows: “rA” is 2′OH A oradenosine; “A” is a 2′-deoxy A or 2′-deoxyadenosine; “mA” is 2′-O-methylA or 2′-methoxy-2′-deoxyadenine; “rG” is 2′-OH G or guanosine; “G” is a2′-deoxy G or 2′-dexoyguanosine; “mG” is 2′-O-methyl G or 2′-methoxy-2′dexoyguanosine; “fC” is 2′-fluoro C or 2′-fluoro-2′ dexoycytidine; “mC”is 2′-O-methyl C or methoxy-2′-dexoycytidine; “fU” is 2′-fluoro U or2′-fluoro-uridine; “mU” is 2′-O-methyl U or 2′-methoxy-uridine; and “iT”is inverted 2′H T, (C6L) is a hexylamino linker; (6GLY) is ahexaethylene glycol spacer; (PEG40KGL2-NOF) is an approximately 40 kDaBranched PEG (SUNBRIGHT™ product No. GL2-400GS2), (6FAM) is6-carboxyfluorescein; (s) is a phosphorothioate linkage between twonucleotides.

Coupling to a Carrier

The GPVI ligands can also include modifications that improvebioavailability or stability. Such modifications can include conjugationto a carrier molecule which may include, but is not limited to ahydrophilic or hydrophobic moiety. One example is polyethylene glycolmolecules conjugated to the nucleic acid sequence. Conjugation to, forexample, a polymer as described below, can confine distribution to theplasma compartment and increase circulating half-life.

Sugar modifications, as described above, can ensure stability but theydo not guarantee adequate pharmacokinetics for nucleic acid ligands tobe therapeutically active. In healthy individuals, ligands are clearedfrom plasma within minutes of IV injection, probably through renalexcretion. Keeping intact ligands in the blood from hours to days afterinjection has been accomplished by conjugating them to largermacromolecules such as polyethylene glycol (PEG). Ligand plasmaclearance has also been decreased by embedding them in liposomes.

Therefore, in one embodiment, the GPVI nucleic acid ligand or GPVIligand modulator can be covalently bound or otherwise attached to anon-immunogenic, high molecular weight compound such as polyethyleneglycol (PEG) or other water soluble pharmaceutically acceptable polymerincluding, but not limited to, polyaminoamines (PAMAM); polysaccharidessuch as dextran, or polyoxazolines (POZ). The GPVI nucleic acid ligandor GPVI ligand modulator can be associated with the high molecularweight compound through covalent bonds. Where covalent attachment isemployed, the high molecular weight compound may be covalently bound toa variety of positions on the ligand or modulator. In some embodiments,the ligand or the modulator can be encapsulated inside a liposome foradministration to a host in need thereof.

In one embodiment, the ligand or modulator is attached to polyethyleneglycol (PEG). Polyethylene glycols (PEGs) can be conjugated tobiologically active compounds to serve as “inert” carriers topotentially (1) prolong the half-life of the compound in thecirculation, (2) alter the pattern of distribution of the compoundand/or (3) camouflage the compound, thereby reducing its immunogenicpotential and protecting it from enzymatic degradation.

The ligand or modulator can attached to the PEG molecule throughcovalent bonds. For example, an oligonucleotide ligand or modulator canbe bonded to the 5′-thiol through a maleimide or vinyl sulfonefunctionality.

Typically, activated PEG and other activated water-soluble polymers areactivated with a suitable activating group appropriate for coupling to adesired site on the therapeutic agent. Representative polymeric reagentsand methods for conjugating these polymers to an active agent are knownin the art and further described in, e.g., Zalipsky, S., et al., “Use ofFunctionalized Poly(Ethylene Glycols) for Modification of Polypeptides”in Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, Plenus Press, New York (1992); and inZalipsky, Advanced Drug Reviews, 1995, 16:157-182. Such reagents arealso commercially available.

For example, in one approach for preparing an amide-linked conjugate, awater soluble polymer bearing an activated ester such as an NHS ester,e.g., mPEG-succinimidyl-α-methylbutanoate, is reacted with an aminegroup of the active agent to thereby result in an amide linkage betweenthe active agent and the water-soluble polymer. Additional functionalgroups capable of reacting with reactive amino groups include, e.g.,N-hydroxysuccinimidyl esters, p-nitrophenylcarbonates,succinimidylcarbonates, aldehydes, acetals, N-keto-piperidones,maleimides, carbonyl imidazoles, azalactones, cyclic imide thiones,isocyanates, isothiocyanates, tresyl chloride, and halogen formates,among others.

In one embodiment, a plurality of GPVI ligands or GPVI ligand modulatorscan be associated with a single PEG molecule. The ligands and modulatorscan be the same or different sequences and modifications. In yet afurther embodiment, a plurality of PEG molecules can be attached to eachother. In this embodiment, one or more GPVI ligands or GPVI ligandmodulators to the same GPVI protein target sequence or different GPVIprotein sequence targets can be associated with each PEG molecule. Inembodiments where multiple ligands or modulators specific for the sametarget are attached to PEG, there is the possibility of bringing thesame targets in close proximity to each other in order to generatespecific interactions between the same targets. Where multiple ligandsor modulators specific for different targets are attached to PEG, thereis the possibility of bringing the distinct targets in close proximityto each other in order to generate specific interactions between thetargets.

While a variety of linkers and methods for conjugation of hydrophilicmoieties such as PEG molecules are well known to persons in the art,several embodiments are provided below. In one embodiment, an aminolinker, such as the C 6 hexylamino linker, 6-(trifluoroacetamido)hexanol(2-cyanoethyl-N,N-diisopropyl)phosphoramidite, shown in FIG. 14, can beused to add the hexylamino linker to the 5′ end of the synthesizedoligonucleotide. Other linker phosphoramidites that may be used to addlinkers to the synthesized oligonucleotides are described below:

TFA-amino C4 CED phosphoramidite (available from ChemGenes, cat#CLP-1453) of the structure:

5′-amino modifier C3 TFA (available from Glen Research cat#10-1923-90)of the structure:

5′-Amino-Modifier C3-TFA

5′-amino modifier 5 (available from Glen Research cat#10-1905-90) of thestructure:

5′-Amino Modifier 5

5′-amino modifier C12 (available from Glen Research cat#10-1912-90) ofthe structure:

5′-Amino-Modifier C12

5′ thiol-modifier C6 (available from Glen Research cat#10-1926-90) ofthe structure:

The 5′-thiol modified linker is used with PEG-maleimides,PEG-vinylsulfone, PEG-iodoacetamide and PEG-orthopyridyl-disulfide, forexample.

The PEG can range in size from 5 to 200 KD, with typical PEGs used inpharmaceutical formulations in the 10-60 KD range. Linear chain PEGs ofup to about 30 KD can be produced. For PEGs of greater than 30 KD,multiple PEGs can be attached together (multi-arm or ‘branched’ PEGs) toproduce PEGs of the desired size. The general synthesis of compoundswith a branched, “mPEG2” attachment (two mPEGs linked via an amino acid)is described in Monfardini, et al., Bioconjugate Chem. 1995, 6:62-69.For ‘branched’ PEGs, i.e. compounds that include more than one PEG ormPEG linked to a common reactive group, the PEGs or mPEGS can be linkedtogether through an amino acid such as a lysine or they can be linkedvia, for example, a glycerine. For branched PEGs in which each mPEG isabout 10, about 20, or about 30KD, the total mass is about 20, about 40or about 60KD and the compound is referred to by its total mass (i.e.40kD mPEG2 is two linked 20 kD mPEGs). 40KD total molecular weight PEGs,that can be used as reagents in producing a PEGylated compound, include,for example, [N²-(monomethoxy 20K polyethylene glycolcarbamoyl)-N⁶-(monomethoxy 20K polyethylene glycol carbamoyl)]-lysineN-hydroxysuccinimide of the structure:

Additional PEG reagents that can be used to prepare stabilized compoundsof the invention include other branched PEG N-Hydroxysuccinimide(mPEG-NHS) of the general formula:

with a 40KD or 60KD total molecular weight (where each mPEG is about 20or about 30KD). As described above, the branched PEGs can be linkedthrough any appropriate reagent, such as an amino acid, and in certainembodiments are linked via lysine residues or glycerine residues.

They can also include non-branched mPEG-Succinimidyl Propionate(mPEG-SPA), of the general formula:

in which mPEG is about 20KD or about 30KD. In a specific embodiment, thereactive ester is —O—CH2CH2-CO2-NHS.

The reagents can also include a branched PEG linked through glycerol,such as the Sunbright™ series from NOF Corporation, Japan. Specific,non-limiting examples of these reagents are:

The reagents can also include non-branched Succinimidylalpha-methylbutanoate (mPEG-SMB) of the general formula:

in which mPEG is between 10 and 30KD. In a subembodiment, the reactiveester is —O—CH₂CH₂CH(CH₃)—CO₂—NHS. Compounds of this structure are soldby Nektar Therapeutics as catalog numbers cat#2M4K0R01.

PEG reagents can also include nitrophenyl carbonate linked PEGs, such asof the following structure:

Compounds of this structure are commercially available, for example fromSunbio, Inc. Compounds including nitrophenyl carbonate can be conjugatedto primary amine containing linkers. In this reaction, the O-nitrophenylserves as the leaving group, leaving a structure[mPEG]_(n)—NH—CO—NH-linker-ligand.

PEGs with thiol-reactive groups that can be used with a thiol-modifiedlinker, as described above, include compounds of the general structure:

in which mPEG is about 10, about 20 or about 30KD. Additionally, thestructure can be branched, such as

in which each mPEG is about 10, about 20, or about 30KD and the totalmass is about 20, about 40, or about 60KD. Branched PEGs with thiolreactive groups that can be used with a thiol-modified linker, asdescribed above, include compounds in which the branched PEG has a totalmolecular weight of about 40 or 60 KD (where each mPEG is 20 or 30 KD).PEG reagents can also be of the following structure:

PEG-maleimide pegylates thiols of the target compound in which thedouble bond of the maleimic ring breaks to connect with the thiol. Therate of reaction is pH dependent and, in one embodiment, is carried outbetween pH 6 and 10, or between pH 7 and 9 or about pH 8.

In one embodiment, a plurality of GPVI ligand modulators can beassociated with a single PEG molecule. The modulator can be to the sameor different GPVI nucleic acid ligands. In embodiments where there aremultiple modulators to the same ligand, there is an increase in aviditydue to multiple binding interactions with the ligand. In yet a furtherembodiment, a plurality of PEG molecules can be attached to each other.In this embodiment, one or more modulators to the same nucleic acidligand or different ligands can be associated with each PEG molecule.This also results in an increase in avidity of each modulator to itstarget.

In one embodiment, the nucleic acid ligand or its modulator can becovalently attached to a lipophilic compound such as cholesterol,dialkyl glycerol, or diacyl glycerol. The lipophilic compound ornon-immunogenic, high molecular weight compound can be covalently bondedor associated through non-covalent interactions with a ligand ormodulator(s). Attachment of the ligand or oligonucleotide modulator tolipophilic or non-immunogenic high molecular weight compounds can bedone directly or with the utilization of linkers or spacers.

In embodiments where direct covalent attachment is employed, thelipophilic compound or non-immunogenic high molecular weight compoundmay be covalently bound to a variety of positions on the ligand ormodulator, such as to an exocyclic amino group on the base, the5-position of a pyrimidine nucleotide, the 8-position of a purinenucleotide, the hydroxyl group of the phosphate, or a hydroxyl group orother group at the 5′ or 3′ terminus.

In embodiments where the ligand or modulator is attached to alipophilic, or a non-immunogenic high molecular weight compound througha linker or spacer, the lipophilic compound or non-immunogenic highmolecular weight compound may be attached to the ligand or modulatorusing, for example, a six carbon amino linker.

In another embodiment, one or more phosphate groups may be includedbetween the linker and the nucleic acid sequence.

Additional suitable linkers and spacers for attaching the ligand ormodulator to a lipophilic compound or to a non-immunogenic highmolecular weight compound are described in U.S. Pat. No. 7,531,524,incorporated herein by reference.

Oligonucleotides of the invention can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve propertiessuch as stability of the molecule and affinity for the intended target.

G. Methods to Treat Platelet-Mediated Disorders

Platelets contain two physiologically important collagen receptors, GPVIand the integrin α₂β₁. Of these, platelet activation in response tocollagen is mediated through GPVI. Activation of platelets by theinteraction of collagen with GPVI leads to release of both dense andalpha-granule contents from platelets. Granule constituents include anumber of platelet agonists as well as pro-inflammatory cytokines,growth factors, adhesion molecules and other proteins including: ATP,GTP, ADP, GDP, polyphosphate, CD63, LAMP2, serotonin, platelet factor 4,β-thromboglobulin, MIP-1α, RANTES, MCP-3, CCL17, CXCL1, CXCL5, IL-8,BFGF, EGF, HGF, insulin-like growth factor 1, TGF-β, VEGF-A, VEGF-C,PDGF, P-Selectin, vWF, thrombospondin, fibrinogen, integrins α_(II)β₃and α_(v)β₃, fibronectin, albumin, α₁-trypsin, Gas6, histidine-richglycoprotein, high molecular weight kininogen, and amyoid beta-proteinprecursor. Thus, platelet activation via the GPVI interaction withcollagen locally creates a pro-inflammatory environment capable ofstimulating a wide range of nearby cell types.

The interaction of GPVI with collagen is also required for plateletadhesion to the diseased or damaged vessel wall. Finally, the activationof GPVI by collagen triggers platelet aggregation. GPVI activation bycollagen plays an important role in hemostasis when vessel damageexposes vascular collagen, thus leading to formation of a platelet plug.While the role of platelets in thrombosis is well understood and ismediated generally by interaction of GPVI with collagen types I and IIIpresent in the sub-endothelial matrix and enriched in atheroscleroticplaques, more recent data indicate that the pro-inflammatory responsesof platelets upon activation can underlie a myriad of disease states inaddition to thrombotic diseases, including atherosclerosis, diabeticvascular disease, rheumatoid arthritis and scleroderma. For example, theinteraction of GPVI with collagen can lead to disease states when theexpression of GPVI is abnormally high, or when platelets are exposed tocollagen at pathophysiologic levels, or when platelets are exposed tocollagen in an abnormal distribution. Accordingly, provided herein aremethods for the treatment of platelet-mediated diseases or disordersusing therapeutically effective amounts of GPVI ligands. These GPVIligands can function by binding to and inhibiting platelet activation.

To understand diseases in which anti-GPVI therapies can produce atherapeutic effect, the specificity of GPVI for the various collagentypes found in the body need only be understood, as platelet activationby collagen requires the collagen be capable of interacting with GPVI.There are 29 different types of collagen. Of these, 9 have beenidentified to be expressed in the vasculature, including types I, III,IV, V, VI, VIII, XII, XIII and XIV. Additionally, of these, 7 arefibrillar and able to assemble into stable triple helices andhigher-order fibrous structures, including types I, II, III, IV, V, XI,XXIV and XXVII (Nieswandt et al., Blood, 2003, 102:449-461; Herr et al.,J. Biol. Chem., 2009, 284:19781-19785). It has been shown that fibrilforming collagens, types I, II, III and IV, support platelet activation,aggregation and adhesion, while the non-fibrillar collagens, types VI,VII and VIII, induce only weak adhesion with no platelet aggregation.Thus, collagen types I-IV can interact with GPVI to specificallyactivate platelets. Additionally, GPVI has been shown to specificallybind collagen types I-IV (Jung et al., Platelets, 2008; 19:32-42). Thus,diseases in which high expression of collagen types I-IV, or in whichabnormal localization or presentation of these collagen types toplatelets occur, can be treated with GPVI ligands.

The structural requirements for GPVI binding by collagen andcollagen-related peptides is well understood (see Example 6; alsoSmethurst et al., J. Biol. Chem., 2007, 282:1296-1304; Smethurst et al.,Blood, 2004, 103:903-911; Horii et al., Blood, 2008:936-942). One of theprimary recognition sites of GPVI within collagen fibrils is thetripeptide sequence GPO. In addition, the collagen binding grooves inGPVI are separated by roughly 5.5 nm. Therefore, other collagen typeswhich contain GPO repeats, and which form fibrillar structures capableof displaying GPO repeats at unit distance of ˜5.5 nm such that they arecapable of binding GPVI dimers, are expected to activate platelets viaGPVI, leading to diseases treatable with GPVI ligands.

GPVI is typically expressed at low to moderate levels on platelets, witha typical receptor number of ˜1200/platelet. However, increased levelsof GPVI expression can make platelets hyper-reactive to collagen, andthus predispose individuals to a disease, or directly cause a diseasestate mediated by the platelet-collagen interaction. Overexpression ofGPVI has been linked to the onset of thrombotic vascular diseases,including transient ischemic attacks (TIA) and acute coronary syndromes(Bigalke et al., Thromb. Res., 2010:125:e184-189; Bigalke et al., Int.J. Cardiol., 2009, Jan. 11 cpub; Bigalke et al., Clin. Res. Cardiol.,2010, 99:227-233). Therefore, treatment of cerebrovascular events suchas TIA and stroke, or treatment of acute coronary syndromes with GPVIligands, can yield a therapeutic effect.

Common molecular mechanisms underlie the role of platelets in hemostasisand inflammatory reactions. For example, in the presence of vascularendothelial damage such as in diabetes and in atherothrombotic vasculardisease, there is a high level of GPVI platelet activation. This resultsin the release of cytokines and activation, localization and maturationof white blood cells. The generation and perpetuation of this process inthe presence of atherosclerotic vascular disease also leads to theadhesion of platelets to the endothelial surface, destabilizing plaquesand leading to further vascular injury. This combination of events canresult in ischemia in the vessel. Thus, a therapeutic such as a GPVIligand, which prevents or reduces unwanted activation of platelets, canbe used to treat a variety of diseases known to be associated with, forexample, upregulation of GPVI expression on platelets or increasedactivation of platelets by increased exposure to collagen.

Diabetes is associated with enhanced collagen-mediated plateletactivation, and GPVI expression is significantly higher in individualswith diabetes as compared to those without (Cabeza et al., Diabetes,2004, 53:2117-2121). High GPVI expression in diabetics contributessignificantly to the thromboischemic complications associated withdiabetes, and thus anti-GPVI therapies can yield a therapeutic effect inthe treatment of diabetic related thromboischemic diseases. Activationof GPVI significantly enhances surface expression of CD40L. CD40L is apotent platelet-derived cytokine involved in thrombosis andatherosclerosis. Furthermore, CD40L overexpression in GPVI-activatedplatelets enhances endothelial surface expression of CD62P, αvβ3,intercellular adhesion molecule 1, and secretion of monocytechemoattractant protein 1. These results indicate that the function ofcollagen receptor GPVI is altered in type 2 diabetes and can play animportant role in diabetic atherothrombotic complications and localizedvascular disease.

Accordingly, GPVI ligands can be used to treat a variety ofplatelet-mediated disorders commonly associated with diabetes. In oneembodiment, a method of treating a subject suffering from diabetes isprovided, comprising administration of a GPVI ligand. Treating high-riskdiabetic patients with GPVI ligands can reduce or preventdiabetes-associated disorders in these patients. These disordersinclude, but are not limited to, diabetic retinopathy, diabeticvasculopathy, atherosclerosis, ischemic stroke, and chronic renalfailure. Treatment of diabetics with GPVI ligands can also reduce orinhibit microthrombus formation in these patients.

Also provided is a method for treating subjects suffering fromplatelet-mediated inflammatory disorders such as rheumatoid arthritis(RA) or other inflammatory arthritis disorders. Recent studies haveshown that people who suffer from RA and other forms of inflammatoryarthritis have increased levels of platelet microparticles in theirjoint fluids (Boilard et al., Science, 2010, 327:580-583). Plateletmicroparticles are pro-inflammatory and elicit an inflammatory responsefrom surrounding cells (eg. synovial fibroblasts). For example, bindingof collagen type IV to GPVI results in the release of IL-1 and IL-8.

Mechanistic studies in mice link the pro-inflammatory platelet state toGPVI activation. The absence of GPVI in knockout mouse models preventsthe recruitment of proinflammatory cells and thus the recruitment andmaturation of white blood cells in the synovium and extracellularmatrix.

Joint inflammation leads to interaction of platelets with collagen injoint extracellular matrix, leading to amplification of inflammation viaGPVI-mediated platelet activation of nearby cells and manifestation ofRA/inflammatory arthritis conditions treatable by GPVI ligands.Accordingly, GPVI ligands can provide therapeutic use in theamelioration, reduction or prevention of inflammatory diseases such asRA or other inflammatory arthritides including, but not limited to,gout, psoriatic arthritis, reactive arthritis, viral or post-viralarthritis and spondylarthritis.

GPVI ligands can also be used to treat subjects suffering fromscleroderma, or systemic sclerosis. Scleroderma appears to occur as anautoimmune response that produces swelling (inflammation) in the musclesand joints, associated with overproduction of collagen. Microvascularinjury is one of the major pathogenic processes involved in systemicsclerosis or scleroderma. Interaction of the platelet type I and IIIcollagen receptor (GPVI) with its respective ligand in the exposedsubendothelial stroma as a result of ongoing microvascular injury insystemic sclerosis patients results in platelet activation andaggregation with the release of pro-inflammatory mediators, whichcontribute to vascular damage and inflammation (Chiang et al.,Thrombosis, 2006, 117:299-306). In systemic sclerosis, vascular lesionsare characterized by an arteriolar capillary perivasculitis withmononuclear cell infiltration that leads to arterial intimalproliferation and obliteration of arterioles and capillaries withattrition of endothelial cells and basal lamina. A recurring pattern ofinjury to the endothelial cells or basal lamina, or both, ischaracteristic of systemic sclerosis. Additionally, these events aredriven by the overproduction and accumulation of collagen in bodytissues, leading to extensive hardening and scarring of tissuesthroughout the body. Accordingly, the use of GPVI ligands can providetherapeutic relief from a disease such as scleroderma or systemicsclerosis which is associated with increased levels of collagen andplatelet-mediated microvascular injury. A method of treating a subjectsuffering from scleroderma by administering a therapeutically effectiveamount of a GPVI ligand is provided herein.

The GPVI ligands disclosed herein can also be used to treat subjectsdiagnosed with cancer. Recent studies suggest that GPVI mediates tumormetastasis (see, e.g., Jain et al., J. Thromb. Haemostasis, 2009,7:1713-1717). Using an in vivo experimental metastasis assay, Jain etal., show that GPVI knockout mice exhibited a significant decrease intumor metastasis as compared to wildtype control mice. Accordingly, inone embodiment, a method for inhibiting, reducing or preventingmetastasis in a subject diagnosed as having a primary cancerous tumor isprovided, wherein the subject is administered a therapeuticallyeffective amount of a GPVI ligand.

Methods, pharmaceutical compositions and uses of the GPVI nucleic acidligands described herein are also provided as modulatable anti-plateletagents for use in disorders or treatment regimes requiring anti-platelettherapy. In certain embodiments, the treatment is a surgicalintervention. The methods can include administering the GPVI nucleicacid ligand to a host in need thereof, wherein the host is sufferingfrom, or at risk of suffering from, an occlusive thrombotic disease ordisorder of the coronary, cerebral or peripheral vascular system.

In one embodiment, the GPVI ligand inhibits initiation of plateletactivation. In other embodiments, the GPVI ligand inhibits plateletactivation and the resultant platelet pro-inflammatory response. Inother embodiments, the GPVI ligand inhibits platelet adhesion. In otherembodiments, the GPVI ligand inhibits platelet aggregation. In yet afurther embodiment, the GPVI ligand inhibits thrombin generation.

In one embodiment, the host has or is at risk of having an occlusivethrombotic disease of the coronary, cerebral and peripheral vascularsystems. In certain other embodiments, the host is preparing to undergoor undergoing a surgical intervention, or has undergone a surgicalintervention that puts the host at risk of an occlusive thromboticevent. In other embodiments, the host has received a vessel graft toenable hemodialysis, which is at risk of occluding due to interactionsbetween the vessel and platelets.

In certain embodiments a method of treating or preventing formation of avascular event, in particular a thrombotic or thromboembolitic event isprovided including administering a GPVI nucleic acid ligand of theinvention to a host in need thereof.

In one embodiment, the GPVI nucleic acid ligand is provided for extendedperiods of time. In this instance, a GPVI ligand modulator may only beused in emergency situations, for example, if treatment leads tohemorrhage, including intracranial or gastrointestinal hemorrhage. Inanother embodiment, the modulator is administered when emergency surgeryis required for patients who have received GPVI nucleic acid ligandtreatment. In another embodiment, the modulator is administered tocontrol the concentration of the GPVI nucleic acid ligand and therebythe duration and intensity of treatment. In another embodiment, the GPVInucleic acid ligand is provided as a platelet anesthetic during acardiopulmonary bypass procedure. In another embodiment, the GPVInucleic acid ligand is administered to provide a period of transitionoff of or on to oral anti-platelet medications, and the modulator isused to reverse the GPVI nucleic acid ligand once therapeutic levels ofthe oral anti-platelet agent are established.

H. Pharmaceutical Compositions

The GPVI nucleic acid ligands or GPVI ligand modulators taught hereincan be formulated into pharmaceutical compositions that can include, butare not limited to, a pharmaceutically acceptable carrier, diluent orexcipient. The precise nature of the composition will depend, at leastin part, on the nature of the ligand and/or modulator, including anystabilizing modifications, and the route of administration. Compositionscontaining the modulator can be designed for administration to a hostwho has been given a GPVI nucleic acid ligand to allow modulation of theactivity of the ligand, and thus regulate anti-platelet activity of theadministered GPVI nucleic acid ligand.

The design and preparation of pharmaceutical or pharmacologicalcompositions will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injection;as tablets or other solids for oral administration; as time releasecapsules, as liquids for oral administration; as elixirs, syrups,suppositories, gels, or in any other form used in the art, including eyedrops, creams, lotions, salves, inhalants and the like. The use ofsterile formulations, such as saline-based washes, by surgeons,physicians or health care workers to treat a particular area in theoperating field may also be particularly useful. Compositions can alsobe formulated for delivery via microdevice, microparticle or sponge.

Pharmaceutically useful compositions comprising a GPVI nucleic acidligand or GPVI ligand modulator of the present invention can beformulated at least in part by the admixture of a pharmaceuticallyacceptable carrier. Examples of such carriers and methods of formulationcan be found in Remington: The Science and Practice of Pharmacy, 20^(th)edition (Lippincott Williams & Wilkins, 2000) and Ansel et al.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 6^(th) Ed.(Media, Pa.: Williams & Wilkins, 1995).

Suitable pharmaceutical excipients include stabilizers, antioxidants,osmolality adjusting agents, buffers, and pH adjusting agents. Suitableadditives include physiologically biocompatible buffers (e.g.,tromethamine hydrochloride), additions of chelants (such as, forexample, EDTA, DTPA or DTPA-bisamide) or calcium chelate complexes (asfor example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additionsof calcium or sodium salts (for example, sodium chloride, calciumchloride, calcium ascorbate, calcium gluconate or calcium lactate).Pharmaceutical compositions of the invention can be packaged for use inliquid form, or can be lyophilized.

To form a pharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofthe nucleic acid ligand or modulator. Such compositions can containadmixtures of more than one compound. The compositions typically containabout 0.1% weight percent (wt %) to about 50 wt %, about 1 wt % to about25 wt %, or about 5 wt % to about 20 wt % of the active agent (ligand ormodulator).

Pharmaceutical compositions for parenteral injectable administration,including subcutaneous, intramuscular or intravenous injections andinfusions are provided herein. For parenteral administration, asepticsuspensions and solutions are desired. Isotonic preparations thatgenerally contain suitable preservatives are employed when intravenousadministration is desired. The pharmaceutical compositions may besterilized and/or contain adjuvants, such as preserving, stabilizing,wetting or emulsifying agents, solution promoters, salts for regulatingthe osmotic pressure and/or buffers. Liquid, particularly injectablecompositions can, for example, be prepared by dissolving, dispersing,etc. The active compound is dissolved in or mixed with apharmaceutically pure solvent such as, for example, water, bufferedwater, saline, 0.4% saline, 0.3% glycine, hyaluronic acid, aqueousdextrose, glycerol, ethanol, and the like, to thereby form theinjectable solution or suspension. Additionally, solid forms suitablefor dissolving in liquid prior to injection can be formulated.

To aid dissolution of an agent into an aqueous environment, a surfactantmight be added as a wetting agent. Surfactants may include anionicdetergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinateand dioctyl sodium sulfonate. Cationic detergents might be used andcould include benzalkonium chloride or benzethomium chloride. Nonionicdetergents that could be included in the formulation as surfactantsinclude, but are not limited to, lauromacrogol 400, polyoxyl 40stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose, carboxymethyl cellulose and any of thepluronic detergents such as Pluronic F68 and/or Pluronic F127 (e.g., seeStrappe et al. Eur. J. of Pharm. Biopharm., 2005, 61:126-133).Surfactants could be present in the formulation of a protein orderivative either alone or as a mixture in different ratios.

For oral administration in the form of a tablet or capsule, the activedrug component can be combined with an oral, non-toxic pharmaceuticallyacceptable inert carrier such as ethanol, glycerol, water and the like.Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents and coloring agents can also be incorporated intothe mixture. Suitable binders include without limitation, starch,gelatin, natural sugars such as glucose or beta-lactose, cornsweeteners, natural and synthetic gums such as acacia, tragacanth orsodium alginate, carboxymethylcellulose, polyethylene glycol, waxes andthe like. Lubricants used in these dosage forms include, withoutlimitation, sodium oleate, sodium stearate, magnesium stearate, sodiumbenzoate, sodium acetate, sodium chloride and the like. Disintegratorsinclude, without limitation, starch, methyl cellulose, agar, bentonite,xanthan gum and the like.

For liquid forms used in oral administration, the active drug componentcan be combined in suitably flavored suspending or dispersing agentssuch as the synthetic and natural gums, for example, tragacanth, acacia,methyl-cellulose and the like. Other dispersing agents that can beemployed include glycerin and the like.

Topical preparations containing the active drug component can be admixedwith a variety of carrier materials well known in the art, such as,e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl ether propionate, and the like, toform, e.g., alcoholic solutions, topical cleansers, cleansing creams,skin gels, skin lotions, and shampoos in cream or gel formulations.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine or phosphatidylcholines. Active agents administereddirectly (e.g., alone) or in a liposomal formulation are described, forexample, in U.S. Pat. No. 6,147,204.

The compounds of the present invention can also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amide-phenol,polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention can be coupled (preferably via a covalent linkage) toa class of biodegradable polymers useful in achieving controlled releaseof a drug, for example, polyethylene glycol (PEG), polylactic acid,polyepsilon caprolactone, polyoxazolines, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels. Cholesteroland similar molecules can be linked to the nucleic acid ligands toincrease and prolong bioavailability.

Lipophilic compounds and non-immunogenic high molecular weight compoundswith which the modulators of the invention can be formulated for use inthe present invention and can be prepared by any of the varioustechniques presently known in the art or subsequently developed.Typically, they are prepared from a phospholipid, for example,distearoyl phosphatidylcholine, and may include other materials such asneutral lipids, for example, cholesterol, and also surface modifierssuch as positively charged (e.g., sterylamine or aminomannose oraminomannitol derivatives of cholesterol) or negatively charged (e.g.,diacetyl phosphate, phosphatidyl glycerol) compounds. Multilamellarliposomes can be formed by the conventional technique, that is, bydepositing a selected lipid on the inside wall of a suitable containeror vessel by dissolving the lipid in an appropriate solvent, and thenevaporating the solvent to leave a thin film on the inside of the vesselor by spray drying. An aqueous phase is then added to the vessel with aswirling or vortexing motion which results in the formation of MLVs. UVscan then be formed by homogenization, sonication or extrusion (throughfilters) of MLV's. In addition, UVs can be formed by detergent removaltechniques. In certain embodiments of this invention, the complexcomprises a liposome with a targeting nucleic acid ligand(s) associatedwith the surface of the liposome and an encapsulated therapeutic ordiagnostic agent. Preformed liposomes can be modified to associate withthe nucleic acid ligands. For example, a cationic liposome associatesthrough electrostatic interactions with the nucleic acid. Alternatively,a nucleic acid attached to a lipophilic compound, such as cholesterol,can be added to preformed liposomes whereby the cholesterol becomesassociated with the liposomal membrane. Alternatively, the nucleic acidcan be associated with the liposome during the formulation of theliposome.

In another embodiment, a stent or medical device may be coated with aformulation comprising a GPVI ligand or GPVI ligand modulator accordingto methods known to skilled artisans.

Therapeutic kits are also envisioned. The kits comprises the reagents,active agents, and materials that may be required to practice the abovemethods. The kits will generally contain, in suitable container means, apharmaceutically acceptable formulation of a GPVI ligand and/or a GPVIligand modulator. The kit may have a single container means, and/or itmay have distinct container means for each compound or each reactionmixture or step.

I. Methods for Administration

Modes of administration of the GPVI ligands and/or GPVI ligandmodulators of the present invention to a host include, but are notlimited to, parenteral (by injection or gradual infusion over time),intravenous, intradermal, intra-articular, intra-synovial, intrathecal,intra-arterial, intracardiac, intramuscular, subcutaneous, intraorbital,intracapsular, intraspinal, intrasternal, topical, transdermal patch,via rectal, vaginal or urethral suppository, peritoneal, percutaneous,nasal spray, surgical implant, internal surgical paint, infusion pump orvia catheter. In one embodiment, the agent and carrier are administeredin a slow release formulation such as an implant, bolus, microparticle,microsphere, nanoparticle or nanosphere. In one embodiment, the GPVInucleic acid ligand is delivered via subcutaneous injection ordeposition including subcutaneous infusion (such as by osmotic pumps).

In one embodiment, the GPVI nucleic acid ligand is delivered viasubcutaneous administration and the modulator is delivered bysubcutaneous or intravenous administration.

The therapeutic compositions comprising ligands and modulators of thepresent invention may be administered intravenously, such as byinjection of a unit dose. The term “unit dose” when used in reference toa therapeutic composition of the present invention refers to physicallydiscrete units suitable as unitary dosage for the host, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requireddiluent; i.e., carrier or vehicle.

Additionally, one approach for parenteral administration employs theimplantation of a slow-release or sustained-released systems, whichassures that a constant level of dosage is maintained.

Local administration, for example, to the interstitium of an affectedjoint, is also provided. Local administration can be achieved byinjection, such as from a syringe or other article of manufacturecontaining a injection device such as a needle. The rate ofadministration from a syringe can be controlled by controlled pressureover desired period of time to distribute the contents of the syringe.In another example, local administration can be achieved by infusion,which can be facilitated by the use of a pump or other similar device.

Representative, non-limiting approaches for topical administration to avascular tissue are also provided and include (1) coating orimpregnating a blood vessel tissue with a gel comprising a nucleic acidligand, for delivery in vivo, e.g., by implanting the coated orimpregnated vessel in place of a damaged or diseased vessel tissuesegment that was removed or by-passed; (2) delivery via a catheter to avessel in which delivery is desired; (3) pumping a composition into avessel that is to be implanted into a patient. Alternatively, thecompounds can be introduced into cells by microinjection, or by liposomeencapsulation.

Also provided is administration of the GPVI ligands to a subject bycoating medical devices such as stents with pharmaceutical compositionscontaining the ligand. Methods for coating to allow appropriate releaseand administration of the ligand are known to those having ordinaryskill in the art.

Optimum dosing regimens for the compositions described herein can bereadily established by one skilled in the art and can vary with themodulator, the patient and the effect sought. The effective amount canvary according to a variety of factors such as the individual'scondition, weight, sex, age and amount of nucleic acid ligandadministered. Other factors include the mode of administration.

Generally, the compositions will be administered in dosages adjusted forbody weight, e.g., dosages ranging from about 1 μg/kg body weight toabout 100 mg/kg body weight. More typically, the dosages will range fromabout 0.1 mg/kg to about 20 mg/kg, and more typically from about 0.5mg/kg to about 10 mg/kg, or about 1.0 to about 5.0 mg/kg, or about 1.0mg/kg, about 2.0 mg/kg, about 3.0 mg/kg, about 4.0 mg/kg, about 5.0mg/kg, about 6.0 mg/kg, about 7.0 mg/kg, about 8.0 mg/kg, about 9.0mg/kg or about 10.0 mg/kg. Typically, the dose initially provides aplasma concentration of drug about 0.002 μg/ml to about 2000 μg/ml ofdrug, more typically from about 2.0 μg/ml to about 400 μg/ml, and moretypically from about 10 μg/ml to 200 μg/ml, or about 20 μg/ml to about100 μg/ml drug, about 20 μg/ml, about 40 μg/ml, about 60 μg/ml, about 80μg/ml, about 100 μg/ml, about 120 μg/ml, about 140 μg/ml, about 160μg/ml, about 180 μg/ml, or about 200 μg/ml.

When administering a modulator to a host which has already beenadministered the ligand, the ratio of modulator to ligand can beadjusted based on the desired level of inhibition of the ligand. Themodulator dose can be calculated based on correlation with the dose ofligand. In one embodiment, the weight-to-weight dose ratio of modulatorto ligand is 1:1. In other embodiments, the ratio of modulator to ligandis greater than 1:1 such as 2:1 or about 2:1, 3:1 or about 3:1, 4:1 orabout 4:1, 5:1 or about 5:1, 6:1 or about 6:1, 7:1 or about 7:1, 8:1 orabout 8:1, 9:1 or about 9:1, 10:1 or about 10:1 or more. In otherembodiments, the dose ratio of modulator to ligand is less than about1:1 such as 0.9:1 or about 0.9:1, 0.8:1 or about 0.8:1, 0.7:1 or about0.7:1, 0.6:1 or about 0.6:1, 0.5:1 or about 0.5:1, 0.45:1 or about0.45:1, 0.4:1 or about 0.4:1, 0.35:1 or about 0.35:1, 0.3:1 or about0.3:1, 0.25:1 or about 0.25:1, 0.2:1 or about 0.2:1, 0.15:1 or about0.15:1, 0.1:1 or about 0.1:1 or less than 0.1:1 such as about 0.005:1 orless. In some embodiments, the ratio is between 0.5:1 and 0.1:1, orbetween 0.5:1 and 0.2:1, or between 0.5:1 and 0.3:1. In otherembodiments, the ratio is between 1:1 and 5:1, or between 1:1 and 10:1,or between 1:1 and 20:1.

GPVI nucleic acid ligands of the present invention can be administeredintravenously in a single daily dose, an every other day dose, or thetotal daily dosage can be administered in several divided doses. Ligandand/or modulator administration may be provide once per day (q.d.),twice per day (b.i.d.), three times per day (t.i.d.) or more often asneeded. Thereafter, the modulator is provided by any suitable means toalter the effect of the nucleic acid ligand by administration of themodulator. Nucleic acid ligands of the present invention can beadministered subcutaneously twice weekly, weekly, every two weeks ormonthly. In some embodiments, the ligands or modulators are administeredless often than once per day. For example, ligand administration may becarried out every other day, every three days, every four days, weekly,or monthly.

In one embodiment, co-administration or sequential administration ofother agents can be desirable. For combination treatment with more thanone active agent, where the active agents are in separate dosageformulations, the active agents can be administered concurrently, orthey each can be administered at separately staggered times.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the host to be treated, capacity of thehost's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are particular to each individual. However, suitable dosage rangesfor systemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for administration are also variable,but are typified by an initial administration followed by repeated dosesat one or more hour intervals by a subsequent injection or otheradministration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in vivo therapies are provided.

EXAMPLES Example 1 Identification of Nucleic Acid Ligands to GPVI

The SELEX method was used to obtain ligands which bind the extracellulardomain of GPVI as described and illustrated in FIG. 1 below.

A starting candidate DNA library was generated by heat annealing andsnap-cooling 1 nmole of template DNA oligo and 1.5 nmoles of 5′ DNAprimer oligo. The sequence of the DNA template oligo for designing thecandidate mixture are: 5′-TCTCGGATCC TCAGCGAGTC GTCTG(N₄₀)CCGCATCGTCCTCCC TA-3′(SEQ ID NO:4) (N₄₀ represents 40 contiguous nucleotidessynthesized with equimolar quantities of A, T, G and C), the 5′ primeroligo and 3′ primer oligo are, respectively, 5′-GGGGGAATTC TAATACGACTCACTATAGGG AGGACGATGC GG-3′ (SEQ ID NO:5) (T7 promoter sequence isunderlined), and 5′-TCTCGGATCC TCAGCGAGTC GTCTG-3′ (SEQ ID NO:6). Thereaction was filled in with Exo⁻ Klenow, stopped by addition of EDTA toa final concentration of 2 mM, and extracted with PCI(phenol:chloroform:isoamyl alcohol (25:24:1)) and then chloroform:isoamyl alcohol (24:1). The extract was desalted, concentrated, andunincorporated nucleotides removed with an Amicon 10 spin column. TheDNA template was utilized in a transcription reaction to generate a2′-fluoropyrimidine starting library. In vitro transcription conditionswere 40 mM Tris-HCl pH 8.0, 4% PEG-800, 12 mM MgCl₂, 1 mM spermidine,0.002% Triton, 5 mM DTT, 1 mM rGTP, 1 mM rATP, 3 mM 2′F-CTP, 3 mM2′F-UTP, 8 μg/mL inorganic pyrophosphatase, 0.5 μM DNA library, andY639F mutant T7 polymerase. Transcriptions were incubated overnight at37° C., DNase treated, chloroform:isoamyl alcohol (24:1) extractedtwice, concentrated with an Amicon 10 spin column, and gel purified on a12% denaturing PAGE gel. RNA was eluted out of the gel, and bufferexchanged and concentrated with TE (10 mM Tris pH 7.5, 0.1 mM EDTA)washes in an Amicon 10 spin column.

The GPVI selection started with a complex library of ˜10¹⁴ different2′-fluoropyrimidine RNA sequences. The complex RNA pool was preclearedagainst a biotin-PEG6-His₆ peptide, immobilized on magnetic streptavidinbeads. The precleared RNA was bound to the purified recombinantextracellular domain of C-term His₆ tagged GPVI protein (SEQ ID NO:3).Purified histidine-tagged GPVI extracellular domain protein was obtainedfrom R&D Systems (Minneapolis, Minn.), Catalog No. 3627-GP, andencompassed residues Gln21-Lys267.

Initial GPVI ligand selection was performed in binding buffer “E,” andstringency was increased in later rounds to binding buffer “F.” Bindingbuffer E consists of 20 mM HEPES pH 7.4, 50 mM NaCl, 2 mM CaCl₂, and0.01% BSA. Binding buffer F consists of 20 mM HEPES pH 7.4, 150 mM NaCl,2 mM CaCl₂, and 0.01% BSA. Protein-RNA complexes were partitioned over a25 mm nitrocellulose disc with washing. The bound RNA was extracted offthe nitrocellulose disc with incubation in PCI (25:24:1). Tris-EDTAbuffer was added and the aqueous phase extracted, followed by achloroform:isoamyl alcohol (24:1) extraction. The resultant bound RNAwas ethanol precipitated. One quarter of the precipitated RNA was heatannealed to the 3′ primer and reverse transcribed utilizing AMV RT. Theentire RT reaction was utilized in PCR with 5′ and 3′ primers andstandard PCR conditions to generate DNA template for the next round ofRNA generation. Specific conditions for each round of selection areshown in FIG. 2. After Round 6, the selection was split into two arms,designated “E2” and “EF.” The E2 selection had Rounds 1-8 performed inbinding buffer E, and Rounds 9-10 performed in binding buffer F. The EFselection had Rounds 1-6 performed in binding buffer E, and Rounds 7-10performed in binding buffer F.

Enrichment of the ligand libraries for GPVI was monitored in directbinding studies utilizing radiolabeled ligand RNA from respective roundsof SELEX and soluble GPVI. Binding studies were performed with trace P³²end-labeled RNA added to serial dilutions of GPVI in Binding Buffer F.To prepare radiolabeled RNAs for binding studies, one hundred picomolesof RNA was dephosphorylated with Bacterial Alkaline Phopshatase at 50°C. for 1 hour. The reaction was phenol: chloroform: isoamyl alcohol(25:24:1) extracted, chloroform: isoamyl alcohol (24:1) extracted, andethanol precipitated. Three pmoles of dephosphorylated RNA was endlabeled with T4 Poylnucleotide Kinase with supplied buffer, and 20 μCiof γ-P³²-ATP and subsequently cleaned with a Biorad MicroBio Spin P-30spin column. End-labeled RNA was diluted to a final concentration of2000 cpm/μL and heat denatured at 65° C. for 5 minutes. RNA and GPVIdilutions were equilibrated at 37° C. prior to use. RNA (5 μL) was addedto varying concentrations of GPVI (15 μL) at 37° C. and incubatedtogether for 5 to 15 minutes. The complexed RNA/GPVI protein mixture wasthen loaded over a Protean BA85 nitrocellulose membrane, overlayed on aGenescreen Plus Nylon membrane, in a 96 well vacuum manifold system withwashing. The membranes were exposed to a phosphorimager screen, scanned,and quantitated with a Molecular Dynamics Storm 840 Phosphorimager. Thefraction bound was calculated by dividing the counts on thenitrocellulose by the total counts and adjusting for the background.Results for binding of enriched ligand libraries from round 10 E2 andround 10 EF of the SELEX as compared to the GPVI-naïve starting ligandlibrary are shown in FIG. 3.

Example 2 Sequencing and Identification of a Structural Family of GPVINucleic Acid Ligands

The final PCR products representing anti-GPVI enriched ligand librariesfrom Round 10 of the SELEX experiments described in Example 1 weredigested with EcoRI and BamH1, cleaned with a purification kit, anddirectionally cloned into linearized pUC19 vector. Bacterial colonieswere streaked for single clones and 5 mL overnight cultures wereinoculated from single colonies. Plasmid DNA was prepared from singlecolonies using Qiagen Plasmid Mini Prep kits. Forty plasmids from eachSELEX experiment were sequenced utilizing a vector primer. DNA sequencesderived from the random region are shown in FIG. 4. Analysis of thesesequences identified 6 unique sequences, which are labeled as “A”through “F” in FIG. 4. The corresponding unique DNA sequences of thefull-length ligand clones are shown in Table 1 below, whereas sequencesrepresenting the random region are provided in Table 2.

TABLE 1 Full-Length DNA Ligands Identified via SELEX Name Sequence GroupSEQ ID NO. EF-1 GGGAGGACGATGCGGCAATCGAAGCTGCATCCAGCGTAAG A 22CCTTCCAGGGATCGTCAGACGACTCGCTGAGGATCCGAGA EF-2GGGAGGACGATGCGGTGCCAAAACACCCGTCTTGCATAAG B 23CCTCCTACGGCAACTCAGACGACTCGCTGAGGATCCGAGA EF-3GGGAGGACGATGCGGATCACACCGCGTCTTGCGTAAGCCT C 24CCTACTAACGGATCGCAGACGACTCGCTGAGGATCCGAGA E2-6GGGAGGACGATGCGGATAGACCGCGTCTGGCATAAGCCTC D 25CAAACACTCTGATCCCAGACGACTCGCTGAGGATCCGAGA EF-22GGGAGGACGATGCGGATTCAACCCGCCTCTGGCATAAGCC E 26TACCCATCGTGATTGTCAGACGACTCGCTGAGGATCCGAGA EF-31GGGAGGACGATGCGGTCTAAGCTGCGTCTGGCATAAGCCT F 27CACCTACTCGATACTCAGACGACTCGCTGAGGATCCGAGA

TABLE 2 Random Regions of A-F Name Sequence Group SEQ ID NO. EF-1CAATCGAAGCTGCATCCAGCGTAAGCCTTCCAGGGATCGT A 11 EF-2TGCCAAAACACCCGTCTTGCATAAGCCTCCTACGGCAACT B 14 EF-3ATCACACCGCGTCTTGCGTAAGCCTCCTACTAACGGATCG C 7 E2-6ATAGACCGCGTCTGGCATAAGCCTCCAAACACTCTGATCC D 15 EF-22ATTCAACCCGCCTCTGGCATAAGCCTACCCATCGTGATTGT E 9 EF-31TCTAAGCTGCGTCTGGCATAAGCCTCACCTACTCGATACT F 12

Representation of these unique ligands as full-length RNA is shown inTable 3, and as RNA sequences indicating the sites of incorporation ofthe 2′-fluorpyrimidine nucleotides used in the SELEX experiment in Table4 (f indicates a 2′-fluorpyrimidine modification and r indicates anon-modified ribonucleotide). Full-length refers to sequences resultingfrom the SELEX process, comprising sequences derived from both therandom portion of the ligand library used in the SELEX process as wellas sequences from the fixed-sequence portions flanking the randomregion.

TABLE 3 Full-Length RNA Sequences Name RNA Sequence Group SEQ ID NO.EF-1- GGGAGGACGAUGCGGCAAUCGAAGCUGCAUCCAGCGUA A 28 RNAAGCCUUCCAGGGAUCGUCAGACGACUCGCUGAGGAUCCG AGA EF-2-GGGAGGACGAUGCGGUGCCAAAACACCCGUCUUGCAUAA B 29 RNAGCCUCCUACGGCAACUCAGACGACUCGCUGAGGAUCCGA GA EF-3-GGGAGGACGAUGCGGAUCACACCGCGUCUUGCGUAAGCC C 30 RNAUCCUACUAACGGAUCGCAGACGACUCGCUGAGGAUCCGA GA E2-6-GGGAGGACGAUGCGGAUAGACCGCGUCUGGCAUAAGCC D 31 RNAUCCAAACACUCUGAUCCCAGACGACUCGCUGAGGAUCCG AGA EF-22- GGGAGGACGAUGCGGAUUCAACCCGCCUCUGGCAUAAGC E 32 RNACUACCCAUCGUGAUUGUCAGACGACUCGCUGAGGAUCCG AGA EF-31- GGGAGGACGAUGCGGUCUAAGCUGCGUCUGGCAUAAGC F 33 RNACUCACCUACUCGAUACUCAGACGACUCGCUGAGGAUCCG AGA

TABLE 4 Full-Length RNA Sequences with Modifications SEQ ID NameModified Sequence* Group NO. EF-1-rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfCrArAfUfCrGrArArGfC A 28 modifiedfUrGfCrAfUfCfCrArGfCrGfUrArArGfCfCfUfUfCfCrArGrGrGrAfUfCrGfUfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCf CrGrArGrA EF-2-rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUrGfCfCrArArArAfCrA B 29 modifiedfCfCfCrGfUfCfUfUrGfCrAfUrArArGfCfCfUfCfCfUrAfCrGrGfCrArAfCfUfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCf CrGrArGrA EF-3-rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfCrAfCrAfCfCrGfCr C 30 modifiedGfUfCfUfUrGfCrGfUrArArGfCfCfUfCfCfUrAfCfUrArAfCrGrGrAfUfCrGfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCf CrGrArGrA E2-6-rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUrArGrAfCfCrGfCrG D 31 modifiedfUfCfUrGrGfCrAfUrArArGfCfCfUfCfCrArArAfCrAfCfUfCfUrGrAfUfCfCfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCf CrGrArGrA EF-22-rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfUfCrArAfCfCfCrGf E 32 modifiedCfCfUfCfUrGrGfCrAfUrArArGfCfCfUrAfCfCfCrAfUfCrGfUrGrAfUfUrGfUfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUf CfCrGrArGrA EF-31-rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUfCfUrArArGfCfUrGfC F 33 modifiedrGfUfCfUrGrGfCrAfUrArArGfCfCfUfCrAfCfCfUrAfCfUfCrGrAfUrAfCfUfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCf CrGrArGrA *SEQ ID NOsrefer to the unmodified versions of the ligands described in the columntitled, “Modified Sequence” rG=2’Ribo G; rA=2’Ribo A; fC=2’-Fluoro C;fU=2’-Fluoro U

Further examination of these six sequences identified a conservedprimary sequence shared by each of the clones consisting of (A/G)UAA)flanked on each side by the sequence GC (eg., GC(A/G)UAAGC), indicatingthese six unique sequences were members of a related family.

Screening of sequences for potential secondary structure was conductedutilizing the mfold server (mfold.bioinfo.rpi.edu). A description ofthese methods is found on the server site as well as in M. Zuker (2003)“Mfold web server for nucleic acid folding and hybridizationprediction.” Nucleic Acids Res. 31 (13), 3406-15 and D. H. Mathews, etal. (1999) “Expanded Sequence Dependence of Thermodynamic ParametersImproves Prediction of RNA Secondary Structure” J. Mol. Biol. 288,911-940. Subsequently, comparative sequence analysis of the uniquesequences was used to align the sequences based upon conserved consensussecondary structural elements to arrive at the predicted secondarystructure of the anti-GPVI ligands (see FIG. 5 and FIG. 8).

The anti-GPVI ligands shared a consensus secondary structure consistingof 3 stems (S1-S3) and 4 loops (L1-L4), with the conserved (A/G)UAAsequence within Loop 4, and the flanking GC sequences forming a pairedregion to comprise Stem 3. Solving the consensus secondary structure ofthe anti-GPVI ligand family identified additional conserved structuralelements, as depicted in FIG. 5. The size and sequence of Loop 1 wasconserved, with Loop 1 consisting of 3 nucleotides of the sequence GAC.The size and sequence of Loop 3 was also conserved, with Loop 3consisting of 2 nucleotides, most often consisting of the sequence UU,or UG, although CA was present in one member of the family. Stem 1consisted of 4-5 basepairs, whereas the length of Stem 2 was longer,typically consisting of 7-8 basepairs. Loop 2 was more variable in size,ranging from 4-7 nucleotides in length.

The affinity of each of the anti-GPVI ligands for GPVI was determined bydirect binding studies using radiolabeled trace ligand RNA and solubleGPVI, per the binding methodology described above in Example 1. Theaffinity of the anti-GPVI ligands for GPVI was high, and ranged from aK_(d) of ˜11 nM to ˜50 nM, with the rank potency of the affinity forGPVI of EF-2≈EF-3≈E2-6≈EF-22≈EF-31>>EF-1. Interestingly, ligand EF-1,which contains the sequence CA in loop 3, exhibited the lowest affinityfor GPVI, consistent with the importance of a loop 3 sequence of UU orUG for high affinity GPVI binding.

Example 3 Truncation and Mutational Probing of Anti-GPVI LigandStructure

The conserved secondary structure of the anti-GPVI ligands presented inFIG. 5 allows reliable predictions as to the minimal sequence requiredto form this structure and bind with high affinity to GPVI. Initialexperiments were aimed at defining the 5′ and 3′ boundary sequencerequirements for maintaining an ligand having the desired structure andfunction.

The consensus secondary structure presented in FIG. 5 indicates thatregions of the ligand derived from the 3′ fixed region of the ligandlibrary are likely dispensable for GPVI binding. The importance of the3′ fixed region derived sequence on interaction with GPVI was tested byheat annealing the complementary DNA 3′ primer onto the P³² end-labeledRNA identified above, equilibrating, and performing binding studies (asdescribed above in Example 1). The results are shown in FIG. 6. Thebinding of the ligand with and without the annealed primer wascomparable, suggesting this region is dispensable for binding to theGPVI protein, as predicted by the consensus structure presented in FIG.5.

Truncated compounds for several of the anti-GPVI ligands containing the5′ and 3′ required sequence boundaries for Stem 1 as predicted by theconsensus structure presented in FIG. 5 were prepared (Table 5), andtheir affinity for GPVI determined. The “RB ID” is a unique identifierthat refers to the ligand having the sequence with specificmodifications noted in the column titled, “Modified Sequence.” The “SEQID NO:” refers to the corresponding nucleic acid sequence (DNA and/orRNA) without modifications.

TABLE 5 GPVI Ligand Truncations and Binding Activity SEQ  Bind- NameID NO: RB ID ing Modified Sequence EF-1 28 RB424 +rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfCrArAfUfCrGrArArGfCfUrGfCrAfUfCfCrArGfCrGfUrArArGfCfCfUfUfCfCrArGrGrGrAfUfCrGfUfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCfCrGrArGrA EF-1 T1 34 RB426 +rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfCrArAfUfCrGrArArGfCfUrGfCrAfUfCfCrArGfCrGfUrArArGfCfCfUfUfCfC EF-2 29 RB427 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUrGfCfCrArArArAfCrAfCfCfCrGfUfCfUfUrGfCrAfUrArArGfCfCfUfCfCfUrAfCrGrGfCrArAfCfUfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCfCrGrArGrA EF-2 T1 35 NA +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUrGfCfCrArArArAfCrAfCfCfCrGfUfCfUfUrGfCrAfUrArArGfCfCfUfCfCfUrAfCrGrGfCrArAfCfU EF-2 T2 36 RB428 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUrGfCfCrArArArAfCrAfCfCfCrGfUfCfUfUrGfCrAfUrArArGfCfCfUfCfCfU EF-2-T2 37 NA NDrGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUrGfCfCrArArArAfCrAfCfCfCrGfUfCr mut3ArArGfCrAfUrArArGfCfCfUfCfCfU EF-2-T2 38 NA NDrGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUrGfCfCrArArArAfCrAfCfCfCrGfUfC mut4fUfUrGfCrAfUfUfUrGfCfCfUfCfCfU EF-2-T2 39 NA NDrGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUrGfCfCrArArArAfCrAfCfCfCrGfUfC mut5fUfUrGfCrAfUrGrGrGfCfCfUfCfCfU EF-3 30 RB429 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfCrAfCrAfCfCrGfCrGfUfCfUfUrGfCrGfUrArArGfCfCfUfCfCfUrAfCfUrArAfCrGrGrAfUfCrGfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCfCrGrArGrA EF-3 T2 40 RB430 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfCrAfCrAfCfCrGfCrGfUfCfUfUrGfCrGfUrArArGfCfCfUfCfCfU EF-3 T3 41 RB445 +rGrGrGrArGrGrAfCrGfUrGfCrGrGrAfUfCrAfCrAfCfCrGfCrGfCfUfUrGfCrGfUrArArGfCfCfUfCfCfC EF-3 T4 42 RB446 +++rGrGrGrArGrGrAfCrGrGfCrGrGrAfUfCrAfCrAfCfCrGfCfCfUfUrGfCrGfUrArArGfCfCfUfCfCfC EF-3 T5 43 RB447 +++rGrGrGrArGrGrAfCrGrGfCrGrAfUfCrAfCrAfCrGfCfCfUfUrGfCrGfUrArArGfCfCfUfCfCfC EF-3 T2 44 NA NDrGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfCrAfCrAfCfCrGfCrGfUfCrArArG mut3fCrGfUrArArGfCfCfUfCfCfU EF-3 T2 45 NA NDrGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfCrAfCrAfCfCrGfCrGfUfCfUfUrG mut4fCrGfUfUfUrGfCfCfUfCfCfU EF-3 T2 46 NA NDrGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfCrAfCrAfCfCrGfCrGfUfCfUfUrG mut5fCrGfUrGrGrGfCfCfUfCfCfU EF-3 T2 47 NA ++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfCrAfCrAfCfCrGfCrGfUfCfUrArG mut6fCrGfUrArArGfCfCfUfCfCfU EF-3 T2 48 NA NDrGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfCrAfCrAfCfCrGfCrGfUfCrAfUrG mut7fCrGfUrArArGfCfCfUfCfCfU EF-3 T5 49 NA +++rGrGrGrArGrGrAfCrGrGfCrGrAfUfCrAfCrAfCrGfCfCfUrGrGfCrGfUrArArG mut8fCfCfUfCfCfC EF-3 T5 50 NA +++rGrGrGrArGrGrAfCrGrGfCrGrAfUfCrAfCrAfCrGfCfCfUfUrGfCrAfUrArArG mut9fCfCfUfCfCfC EF-3 T5 51 NA +++rGrGrGrArGrGrAfCrGrGfCrGrAfUfCrAfCrAfCrGfCfCfUrGrGfCrAfUrArAr mut10GfCfCfUfCfCfC E2-6 31 RB431 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUrArGrAfCfCrGfCrGfUfCfUrGrGfCrAfUrArArGfCfCfUfCfCrArArAfCrAfCfUfCfUrGrAfUfCfCfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCfCrGrArGrA E2-6 T1 52 RB432 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUrArGrAfCfCrGfCrGfUfCfUrGrGfCrAfUrArArGfCfCfUfCfC EF-22 32 RB433 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfUfCrArAfCfCfCrGfCfCfUfCfUrGrGfCrAfUrArArGfCfCfUrAfCfCfCrAfUfCrGfUrGrAfUfUrGfUfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCfCrGrArGrA EF-22 T1 53 RB434 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGrAfUfUfCrArAfCfCfCrGfCfCfUfCfUrGrGfCrAfUrArArGfCfCfUrAfCfCfC EF-31 33 RB435 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUfCfUrArArGfCfUrGfCrGfUfCfUrGrGfCrAfUrArArGfCfCfUfCrAfCfCfUrAfCfUfCrGrAfUrAfCfUfCrArGrAfCrGrAfCfUfCrGfCfUrGrArGrGrAfUfCfCrGrArGrA EF-31 T1 54 RB436 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUfCfUrArArGfCfUrGfCrGfUfCfUrGrGfCrAfUrArArGfCfCfUfCrAfCfCfUrAfCfU EF-31 T2 55 RB439 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUfCfUrArArGfCfUrGfCrGfUfCfUrGrGfCrAfUrArArGfCfCfUfCrAfCfC EF-31 T3 56 RB440 +++rGrGrGrArGrGrAfCrGrAfUrGfCrGrGfUfCfUrArArGfCfUrGfCrGfUfCfUrGrGfCrAfUrArArGfCfCfUfCfCfC EF-31 T4 57 RB441 +rGrGrGrArGrGrAfCrGfUrGfCrGrGfUfCfUrArArGfCfUrGfCrGfCfUrGrGfCrAfUrArArGfCfCfUfCfCfC EF-31 T5 58 RB442 +++rGrGrGrArGrGrAfCrGrGfCrGrGfUfCfUrArArGfCfUrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfC EF-31 T6 59 RB443 +++rGrGrGrArGrGrAfCrGrGfCrGfUfCfUrArArGfCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfC EF-31 T7 60 RB444 +++rGrGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUrGrGfCrAfUrArArGf CfCfUfCfCfCEF-31 T8 61 NA +rGrGrGrArGrGrAfCrGrGfCfCfUrArArGfCfCfUrGrGfCrAfUrArArGfC fCfUfCfCfC AllRNAs were in vitro transcribed from either PCR generated or Klenowgenerated templates SEQ ID NOs refer to the unmodified versions of theligands described in the column titled, “Modified Sequence” rG=2’Ribo G;rA=2’Ribo A; fC=2’-Fluoro C; fU=2’-Fluoro U Underlined nucleotides noteintroduced changes ++++ K_(d) less than 10 nM +++ K_(d) l0 nM-24 nM ++K_(d) 25 nM-40 nM + K_(d)>40 nM ND Binding not detected NA Not Assigned

Binding data for several representative truncates are shown in FIG. 7.Consistent with the consensus 5′ and 3′ boundaries predicted by thesecondary structure defined in FIG. 5, EF-1 T1, EF2 T2, EF-3 T2, E2-6T1, EF-22 T1, and EF-31 T2 bound GPVI with a K_(d) equivalent to therespective full-length parent ligand, confirming the predictedboundaries of the 5′ and 3′ end are sufficient to form Stem 1 andfunctional anti-GPVI ligands (See Table 5).

The consensus structure presented in FIG. 5 suggests that Stem 2 maycontain more basepairs than are required to form a stable stemstructure. Furthermore, the conservation of the size and sequence ofLoop 1, which is derived from the 5′ fixed region of the starting ligandlibrary, combined with the size of Stem 2 and lack of conservation inthe length or sequence of Loop 2, suggest that Stem 2 and Loop 2 mayhave, in part, a spacing function to properly present Loop 1. Based onthese insights, it may be possible to reduce the size of Stem 2 whileretaining high affinity binding to GPVI. Therefore, additional truncateswere prepared to determine the minimal size of Stem 2 required for highaffinity binding to GPVI (See FIGS. 8A, B, C for secondary structurerepresentations and Table 5 for sequence identifiers). EF-3 T4 and T5,and EF-31 T5, T6 and T7 bound GPVI with a K_(d) equivalent to therespective full-length parent ligand, confirming that a Stem 2 as shortas 4 basepairs in length is sufficient for high affinity binding toGPVI. These results are entirely consistent with a role for Stem 2 andLoop 2 in properly presenting the GAC sequence of Loop 1. Additionally,truncates EF-3 T3 and EF-31 T4, which deleted the A-U basepair at the2^(nd) position from the base of Stem 2, both exhibited reduced bindingas compared to the respective full-length parent ligand. Examination ofthe Stem 2 composition for these truncates as compared to the Stem 2composition for the truncates listed above, which did not exhibitreduced GPVI affinity, revealed that truncates EF-3 T3 and EF-31 T4uniquely positioned a U-G at this position within Stem 2, as opposed toa G-C basepair or the A-U basepair present in the parental sequences.This indicates that a purine-pyrimidine pair may be required at the2^(nd) basepair from the base of Stem 2 for high affinity GPVI binding,a feature conserved in the consensus secondary structure of theanti-GPVI ligands presented in FIG. 5.

The consensus secondary structure of the anti-GPVI ligands presented inFIG. 5 shows primary sequence conservation within Loops 1, 3 and 4, andno conservation in the size or sequence content of Loop 2. Mutations andsubstitutions were constructed within these loops to evaluate thefunctional importance of the observed conservation, or lack thereof, inthese loop sequences (see Table 5 above; the underlined portionsindicate mutated residues).

The conserved UU found within Loop 3 of EF-2 T2 or EF-3 T2 was mutatedto AA (EF-2 T2mut3 and EF-3 T2mut3, See FIG. 9 for secondary structurerepresentations), resulting in complete loss of measurable binding toGPVI, consistent with the importance of a UU or UG sequence for Loop 3as predicted by the consensus secondary structure presented in FIG. 5.The conserved UU located within Loop 3 in EF-3 T2 was also mutated to UA(EF-3 T2mut6) and AU (EF-3 T2mut7). Consistent with the consensusstructure of the anti-GPVI ligands, mutation of the first U of Loop 3 toA resulted in loss of measurable binding to GPVI, while mutation of thesecond U of Loop 3 to A resulted in a slight decrease in affinity forGPVI. Thus, a Loop 3 sequence of UA, like UU or UG, supports highaffinity binding of anti-GPVI ligands to GPVI, and is consistent withthe observed conservation of Loop 3 as 5′-YD-3′, where Y represents apyrimidine and a U is highly favored as the pyrimidine in Loop 3, and Drepresents U or G or A (not C).

The conserved GUAA of EF-3 T2 and AUAA of EF-2 T2 within Loop 4 weremutated to GUUU (EF-3 T2mut4) and AUUU (EF-2 T2mut4) respectively, aswell as to GUGG (EF-3 T2mut5) and AUGG (EF-2 T2mut5) (See FIG. 9 forsecondary structure representations). Both sets of substitutionsresulted in complete loss of measurable GPVI binding, consistent withthe importance of the Loop 4 sequence of AUAA or GUAA to GPVI binding aspredicted by the consensus secondary structure presented in FIG. 5. Tofurther verify the importance of the conserved sequences within Loops 3and 4, Loop 3 of EF-3 T2 was converted to UG (EF-3 T2mut8), as found inthe EF-22, E2-6 and EF-31 sequences, which resulted in no significantchange in affinity for GPVI, consistent with the conserved structure ofthe anti-GPVI ligands presented in FIG. 5. Additionally, Loop 4 of EF-3T2 was converted to AUAA (EF-3 T2 mut9) as found in EF-2, EF-22, E2-6and EF-31, which resulted in no significant change in affinity for GPVI,consistent with the conserved structure of the anti-GPVI ligandspresented in FIG. 5. Finally, simultaneous conversion of EF-3 T2 Loop 3to UG and Loop 4 to AUAA (EF-3 T2mut10), as found in EF-22, E2-6 andEF-31, resulted in no significant change in affinity for GPVI,consistent with the conserved structure of the anti-GPVI ligandspresented in FIG. 5.

Synthetic anti-GPVI ligands with single-point mutations in Loop 1(RB470-RB472, see Table 6 below) were produced, converting the Loop 1sequence to CAC, GUC, and GAG respectively. Each substitution resultedin complete loss of measurable GPVI binding, consistent with theimportance of the Loop 1 sequence of GAC to GPVI binding as predicted bythe consensus secondary structure presented in FIG. 5.

Loop 2 exhibited no apparent conservation in length or sequencecomposition, but rather, may have served a role as a spacer in theisolated ligands, to enable presentation within Loop 1 of the GACsequence derived from the 5′ fixed sequence of the ligand library usedin SELEX. This would predict that a nucleotide composition of Loop 2 isnot required for high affinity binding to GPVI. Consistent with thisprediction, substitution of Loop 2 of EF-31 T7 (RB466) with ahexaethylene glycol spacer (See FIGS. 13A-B) resulted in no loss ofaffinity for GPVI, as compared to the parent ligand (RB448). Bindingdata for RB 466 are presented in FIG. 10.

Example 4 Further Truncation and Optimization of the 2′ SugarModification and Phosphodiester Backbone of Anti-GPVI Ligand

Ligands isolated from 2′-fluorpyrimidine/2′-hydroxypurine librariesexhibit sufficient nuclease stability for in vitro screening. However,the high 2′-hydroxyl content make them unsuitable for drug developmentcandidates due to the fact that these positions can be very sensitive tonuclease degradation in vivo, limiting the maximal concentration thatcan be achieved post parenteral administration as well as theircirculating half-life. Therefore, we sought to optimize the anti-GPVIligands by further stabilization of the backbone by substitution of2′-O-methyl nucleotides for 2′-hydroxyl nucleotides, or by substitutionof 2′-deoxy nucleotides for 2′-hydroxyl nucleotides, with modificationof the ligand backbone by phosphorothioate substitution as needed topreserve affinity for GPVI while enhancing nuclease stability.Additional substitutions of 2′-O-methyl nucleotides for 2′-fluoronucleotides were also made to further improve stability, reduce cost ofmanufacturing, and reduce the level of potential impurities that canarise during heating of 2′-fluorouridine-containing oligonucleotidesduring manufacturing processes. Finally, “capping” of the 5′ and 3′ends, which prevents exonucleasc degradation of oligonucleotides, wasalso attempted to further enhance in vivo stability. Conservation of theminimal required sequence amongst the anti-GPVI ligands was quite high,and in general, their affinities for GPVI were similar. Ligand EF-31 T7represented a minimal anti-GPVI ligand with high GPVI affinity and areduced length of Loop 2, and was therefore chosen as a parent moleculefor further optimization of the anti-GPVI ligands for nuclease stabilityand GPVI binding.

Capping of the 3′ end of EF-31 T7 was accomplished by synthesis of theligand from a CPG-support loaded with inverted deoxythymidine, to createa 3′-3′ linkage (RB448) at the 3′ end of the ligand. This modificationwas well tolerated, and was therefore used in all synthetically producedmodifications to this ligand. RB448 (SEQ ID NO:62) has the sequence:

rGrGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiT,wherein “r” represents a ribonucleic acid, “f” represents a 2′-fluoronucleotide, and “iT” represents the inverted deoxythymidine.

Initial substitutions of 2′Omethyl nucleotides for 2′ hydroxyl and 2′fluoro nucleotides were synthesized within Stems 1, 2 and 3, and testedfor binding to GPVI in direct binding assays as described in Example 1.Results of the binding assays are presented in Table 6 below.Substitution of most (RB452) or all (RB453) of the 2′-hydroxyls with2′-O-methyls within Stem 1 was well tolerated, with an all 2′-O-methylcomposition of Stem 1 resulting in an increase in affinity for GPVI ascompared to the parent compound (RB448), consistent with the conservedsecondary structure of the anti-GPVI ligands. Substitution of the2′-hydroxyls with 2′-O-methyls within the upper 2 basepairs of Stem 2(RB455) was also well tolerated, consistent with the conserved secondarystructure and truncation data, which indicates several combinations ofbasepair modifications in the upper portion of Stem 2 can be presentwithin an anti-GPVI ligand and support high affinity binding to GPVI.Finally, substitution of the 2′-hydroxyl-2′-fluoro G-C basepair with a2′-O-methyl G-C basepair in Stem 3 (RB462) resulted in a significantincrease in affinity for GPVI. This substitution is expected to increasethe stability of Stem 3, and is consistent with the consensus secondarystructure of the anti-GPVI ligands, in which Stem 3 is predicted to be ashort, 2 basepair stem.

A composite molecule consisting of the substitutions within RB453,RB455, RB462 and RB466 was next synthesized (RB490) and tested forbinding to GPVI in direct binding assays (See FIG. 10). Consistent withthe affinity of compositions containing the individual sets ofsubstitutions, RB490 bound GPVI with significantly greater affinity thanthe starting parent compound, with a K_(d) for GPVI of ˜4-5 nM ascompared to ˜14-15 nM for RB448. Subsequently, RB490 served as theparent compound for further optimization of the anti-GPVI ligand. Itshould be noted that, although the 2′-O-methyl substitution at the firstresidue of the molecule (G at position 1) is well tolerated, thismodification greatly reduces the efficiency of 5′ end-labeling of thesecompositions with P³², and therefore this residue was left as a2′-hydroxyl for the evaluation of the majority of the remainingmodifications tested to facilitate direct binding studies. Secondarystructure representations noting the sugar modifications for keycomposite compositions discussed within can be found in FIG. 11A-D.

Because the 5′ half of Stem 2 is derived from the 5′ fixed region of theligand library used in the SELEX studies, the sequence composition ofthe stem sequence may be a less reliable guide for the likelihood oftolerability of substitutions for the 2′-hydroxyl or 2′-fluoronucleotides in the bottom two basepairs of this stem than in sequencesderived from the random region of the ligand library. Therefore,individual 2′-O-methyl for 2′-hydroxyl and 2′-fluoro substitutions weresynthesized for the two G-C basepairs at the base of Stem 2 (RB497, 498,499, and 500). 2′-O-methyl for 2′-hydroxyl substitutions were welltolerated for the G in the G-C basepair at the terminal position of Stem2 (RB497), but the 2′-O-methyl for 2′ fluoro substitution for the C ofthis basepair (RB498) was not well tolerated. Substitution of2′-O-methyl for 2′-hydroxyl and for 2′-fluoro sugars was well toleratedfor both the G and C in the G-C basepair in the second position of Stem2 (RB499 and RB500), although a modest reduction in affinity was notedfor the 2′-O-methyl substitution for the G (RB499).

Several of the consensus secondary structure predictions for theanti-GPVI ligands suggested that Stem 1 could be as short as 4basepairs. Consistent with the consensus secondary structure, deletionof one of the G-C basepairs within Stem 1 was well-tolerated (RB507),with no loss of affinity as compared to the parental composition RB490.

Modification of the first G-C basepair within Stem 3 to a basepaircontaining 2′-O-methyl sugars (RB462) resulted in increased affinity ascompared to a composition with a 2′-hydroxl-2′-fluoro G-C basepair atthat position. In contrast, 2′-O-methyl substitution of the sugars inthe C-G basepair within Stem 3 resulted in loss of measurable GPVIaffinity (RB463). Therefore, to further investigate the optimal sugarmodification pattern for Stem 3, single substitutions of 2′-O-methyl for2′-fluoro and 2′-hydroxyl sugars were made, respectively, for the C-Gbasepair of Stem 3 (RB501 and RB502). Substitution of a 2′-O-methyl for2′-hydroxyl sugar in the G of this C-G basepair was well tolerated, withno apparent loss of affinity for GPVI (RB502), whereas the 2′-O-methylfor 2′-fluoro substitution in the C of this C-G basepair resulted inloss of measurable GPVI affinity (RB501), indicating this substitutionwithin RB463 was the cause of the loss of GPVI binding observed withthis composition. Overall, the preferred sugar modification pattern ofStem 2 is consistent with the anti-GPVI ligand consensus structure, andindicates the presence of the C within this C-G basepair of Stem 3 isconserved at the level of the sugar modification, stem formation, andprimary sequence.

The sequence of Loop 4 is highly conserved within the anti-GPVI ligands,and therefore individual 2′-O-methyl substitutions were made for thesugars at each of these positions within Loop 4 (RB503, RB504, RB505 andRB506). 2′-O-methyl substitutions were well tolerated at each of thesepositions, with substitution of 2′-O-methyl for 2′-hydroxyl sugars ateach A within Loop 4 yielding compositions with affinity for GPVIequivalent to the parent composition RB490 (RB503, RB505 and RB506),whereas the 2′-O-methyl for 2′-fluoro sugar substitution at the U withinLoop 4 (RB504) resulted in a modest loss of GPVI affinity. Nonetheless,because the 2′-fluoro sugar modification affords significant nucleaseresistant, the U within this loop was maintained as a 2′-fluoronucleotide.

Mutational analysis (See Example 3, RB470, RB471 and RB472) demonstratedthe importance of the GAC sequence within Loop 1 for high affinitybinding to GPVI. Therefore, individual 2′-O-methyl and 2′-deoxysubstitutions for the sugars at each of the positions within Loop 1 weresynthesized to determine the preferred sugar modification pattern of theLoop 1 nucleotides (RB491, RB492, RB493, RB494, RB495, and RB496).2′-O-methyl for 2′-hydroxyl substitution of the G within Loop 1 was welltolerated (RB491), within an affinity for GPVI equivalent to theparental composition RB490, and subsequently the 2′-deoxy substitutionat this position was not tested (RB494). Substitution of 2′-deoxy for2′-hydroxyl at the A within Loop 2 (RB495) was better tolerated thansubstitution of a 2′-O-methyl at this position (RB492), with an affinityfor GPVI approximately equivalent to the parental composition RB490.Substitution of either 2′-O-methyl or 2′-deoxy sugars for the 2′-fluorosugar of the C within Loop 2 resulted in modest loss of GPVI affinity,and therefore was not further pursued. Consistent with individual sugarsubstitutions within Loop 1, a composition with Loop 1 consisting of2′-O-methyl G, 2′-deoxy A, and 2′-fluoro C (RB519) bound GPVI withhigher affinity than a composition with Loop 1 consisting of 2′-O-methylG, 2′-O-methyl A, and 2′-fluoro C (RB518).

Composite molecules consisting of substitutions within RB491, RB502,RB503, RB505 and RB506 (RB520) and consisting of substitutions withinRB491, RB502, RB503, RB505, RB506 and RB507 (RB521) were synthesized toverify that incorporation of each of these substitutions within a singlecomposition was well tolerated. As expected from the individualsubstitutions, RB520 and RB521 (same sugar substitution pattern as RB520but with a 4 basepair Stem 1), bound GPVI with a IQ similar to bindingof RB490 to GPVI (approximately 6 nM vs 4-5 nM for RB490). Similarly, acomposite consisting of these substitutions, but with a 2′-fluoro at the2^(nd) G-C basepair of Stem 2 (RB524) also bound GPVI with highaffinity.

RB524 contained 4 remaining 2′ hydroxyl residues within the central coreof the molecule (defined by the top of Stem 1, Loop 1, the base of Stem2, Loop 3, Stem 3, and Loop 4). To determine optimal sugar substitutionpatterns for the remaining 2′-hydroxyl residues in the context of thehighly modified RB524 composition, single, pair-wise and multiple sugarsubstitutions previously studied independently were synthesized in theRB524 background. Inclusion of a Loop 1 sequence of 2′-O-methyl G,2′-deoxy A and 2′-fluoro C in conjunction with a 2′-deoxy substitutionat the G in the terminal G-C basepair of Stem 2 into the RB524background (RB526) resulted in a composition which retained highaffinity binding to GPVI, with only two remaining 2′-hydroxyls.Subsequently, as expected from previous analysis of the length of Stem1, deletion of a G-C basepair within the RB526 context (RB537) resultedin a composition that maintained high affinity GPVI binding.Substitution of a 2′-O-methyl for the 2′-hydroxy sugar at the G of Loop3 (RB538) in the 537 background also resulted in a composition thatmaintained high affinity GPVI binding. Finally, substitution of a2′-deoxy for the lone 2′-hydroxyl remaining at the G of the 2^(nd) G-Cbasepair within Stem 2 of RB538 (RB540) was well tolerated, yielding acomposition fully-substituted at the 2′ position of the sugars with aK_(d) for GPVI of approximately 15 nM.

Functional studies (See Example 6) indicated that there was a goodcorrelation between affinity for GPVI and inhibition of collagen orCRP-induced platelet aggregation. Additionally, the ability of theanti-GPVI ligands to block CRP-induced platelet aggregation suggestedthat the ligands may bind to a region of the collagen binding domainoverlapping or near to the CRP-binding site on GPVI. The collagenbinding site on GPVI has a net positive charge, and a largeelectropositive surface. Therefore, phosphorothioate substitutions weremade within RB540 in the regions consisting of 2′-deoxy substitutionswithin Loop 1 and Stem 2, as phosphorothioate substitutions increase thenet electronegativity of oligonucleotides, and thereby may lead toincrease affinity of the ligands for GPVI. Additionally,phosphorothioate substitutions in the context of 2′-deoxysugar-containing residues may afford additional protection from nucleasedegradation. Individual phosphorothioate substitutions were madeflanking the A and C of Loop 1, and the G's of the terminal 2 basepairsof Stem 2 (RB546, RB547, RB548, RB549, RB550) in the RB540 background.Substitution of single phosphorothiates between the two G's at the baseof Stem 2 (RB549) or 3′ to the 2^(nd) G of Stem 2 (RB550) enhanced theaffinity of the ligands for GPVI. Inclusion of both of thesephosphorothioate substitutions into a composite anti-GPVI ligand (RB566)further enhanced the affinity of the ligand for GPVI, resulting in anligand exhibiting a slightly enhanced affinity for GPVI as compared toRB490, with a K_(d) for GPVI of approximately 2-4 nM, and a greatlyenhanced affinity as compared to RB540 (see FIG. 12).

In addition to the extent of nuclease stabilization, distribution andhalf-life of ligands post parenteral administration is greatly impactedby their molecular weight. Conjugation of ligands to high molecularweight carriers, such as high molecular weight polyethylene glycol(PEG), limit the distribution of an ligand to mainly the plasmacompartment, leading to higher C_(max) per dose unit, and greatly limitrenal filtration of the ligand, and thus greatly enhance the ligand's invivo potency and circulating half-life. Given that a control agent maybe administered to finely tune the potency and half-life of an anti-GPVIligands (see Example 8), anti-GPVI ligands were conjugated to a highmolecular weight carrier to provide the greatest potential half-lifewith distribution mainly limited to the plasma compartment. PEGylationof ligands can be achieved by conjugation of the PEG to a unique site onthe ligand, added by incorporation of a site-specific linker to theligand during synthesis. Therefore, the impact of linker addition andPEG conjugation to RB540, RB549 and RB566 was assessed. 5′hexylamino-linker containing compositions of these respective ligands(RB542, RB560 and RB567) bound GPVI with affinities equivalent to thenon-linker containing parent compositions. It should be noted that, forcompositions containing either a 5′ linker or a 5′ linker and PEG, thesugar at G of position 1 of the ligand was 2′-O-methyl. Additionally, asincorporation of a linker to the 5′ end of the ligand prevents labelingof the 5′ end with P³², affinity of linker and PEG modified ligands wasdone via competition binding against the cognate radiolabeled parentligand, using essentially the methods outlined in Example 1. Conjugationof a branched 40KDa PEG to the respective linker containing ligands(RB569, RB570, RB571) resulted in an approximately 1.2 to 1.7 folddecrease in GPVI affinity as compared to the non-linker, or linker only,containing parent compositions (eg. RB566 for RB571). Thus, RB571, whichconsists of the RB566 composition (with a 2′-O-methyl G at position 1)conjugated to a 40KDa PEG, exhibits a K_(d) for GPVI of approximately 5nM.

TABLE 6 Modified GPVI Ligands Bind- SEQ  RB ID Modified Sequence ingID NO RB448rGrGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiT+++ 62 RB450mGmGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCmCmCiTND 62 RB451mGmGmGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUrGrGfCrAfUrArArGfCfCfUmCmCmCiTND 62 RB452mGmGmGmArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUrGrGfCrAfUrArArGfCfCmUmCmCmCiT+++ 62 RB453mGmGmGmAmGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUrGrGfCrAfUrArArGfCmCmUmCmCmCiT++++ 62 RB454rGrGrGrArGmGmAmCrGrGfCrGfCfUrArAfCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiTND 62 RB455rGrGrGrArGrGrAfCrGrGmCmGfCfUrArAmCmGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiT+++ 62 RB456rGrGrGrArGrGrAfCrGmGmCmGfCfUrArAmCmGmCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiTND 62 RB459rGrGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCmUrGrGfCrAfUrArArGfCfCfUfCfCfCiTdead 62 RB460rGrGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUmGrGfCrAfUrArArGfCfCfUfCfCfCiT+++ 62 RB461rGrGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCmUmGrGfCrAfUrArArGfCfCfUfCfCfCiTdead 62 RB462rGrGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUrGmGfCrAfUrArArGmCfCfUfCfCfCiT++++ 62 RB463rGrGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCfUrGrGmCrAfUrArAmGfCfCfUfCfCfCiTdead 62 RB466rGrGrGrArGrGrAfCrGrGfCrG(6GLY)fCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiT+++ 63 RB469(6FAM)rGrGrGrArGrGrAfCrGrGfCrGfCfUrArAfCrGfCfCrArArGfCrAfUrArArGfCfCfUfCdead 64 fCfCiT (**) RB470rGrGrGrArGfCrAfCrGrGfCrG(6GLY)fCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiTdead 65 RB471rGrGrGrArGrGfUfCrGrGfCrG(6GLY)fCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiTdead 66 RB472rGrGrGrArGrGrArGrGrGfCrG(6GLY)fCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiTdead 67 RB473mGrGrGrArGrGrAfCrGrGfCrG(6GLY)fCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiTND 63 RB478(6FAM)rGrGrGrArGrGrAfCrGrGfCrG(6GLY)fCrGfCfCfUrGrGfCrAfUrArArGfCfCfUfC+++ 63 fCfCiT (**) RB480rGrGrGrArGrGrAfCrGrGfCrG(6GLY)fCrGfCfCfUfUrGfCrAfUrArArGfCfCfUfCfCfCiT+++ 68 RB482rGrGrGrArGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGrGfCrAfUrArArGfCfCfUfCfCfCiTND 63 RB485mGmGmGmAmGrGrAfCrGrGfCrG(6GLY)fCrGfCfCfUrGrGfCrAfUrArArGfCmCmUmCmCmCiTND 63 RB487mGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGrGfCrAfUrArArGfCmCmUmCmCmCiTND 63 RB488rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGrGfCrAfUrArArGfCmCmUmCmCmCiT++++ 63 RB490rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT++++ 63 RB491rGmGmGmAmGmGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT++++ 63 RB492rGmGmGmAmGrGmAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT+++ 63 RB493rGmGmGmAmGrGrAmCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT+++ 63 RB494rGmGmGmAmGGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT ND63 RB495rGmGmGmAmGrGAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT++++ 63 RB496rGmGmGmAmGrGrACrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT+++ 63 RB497rGmGmGmAmGrGrAfCmGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT++++ 63 RB498rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCmCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT++ 63 RB499rGmGmGmAmGrGrAfCrGmGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT+++ 63 RB500rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGmCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT++++ 63 RB501rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGmCrAfUrArArGmCmCmUmCmCmCiTdead 63 RB502rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArAmGmCmCmUmCmCmCiT++++ 63 RB503rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCmAfUrArArGmCmCmUmCmCmCiT++++ 63 RB504rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAmUrArArGmCmCmUmCmCmCiT+++ 63 RB505rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUmArArGmCmCmUmCmCmCiT++++ 63 RB506rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrAmArGmCmCmUmCmCmCiT++++ 63 RB507rGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCiT ++++69 RB508(C6L)rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmC++++ 63 mCmCiT RB509rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCAfUrArArGmCmCmUmCmCmCiT ND63 RB510rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrATrArArGmCmCmUmCmCmCiT ND70 RB511rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUArArGmCmCmUmCmCmCiT ND63 RB512rGmGmGmAmGrGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrAArGmCmCmUmCmCmCiT ND63 RB517rGmGmGmAmGmGmAmCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT +63 RB518rGmGmGmAmGmGmAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT+++ 63 RB519rGmGmGmAmGmGAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCrAfUrArArGmCmCmUmCmCmCiT++++ 63 RB520rGmGmGmAmGmGrAfCrGrGmCmG(6GLY)mCmGmCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT++++ 63 RB521rGmGmAmGmGrAfCrGrGmCmG(6GLY)mCmGmCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCiT ++++69 RB522rGmGmGmAmGmGmAfCrGrGmCmG(6GLY)mCmGmCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT++ 63 RB523rGmGmGmAmGmGmAfCmGrGmCmG(6GLY)mCmGmCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT +63 RB524rGmGmGmAmGmGrAfCrGrGmCmG(6GLY)mCmGfCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT+++ 63 RB525rGmGmGmAmGmGAfCmGrGmCmG(6GLY)mCmGfCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT ++63 RB526rGmGmGmAmGmGAfCGrGmCmG(6GLY)mCmGfCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT +++63 RB527rGmGmGmAmGmGmAfCmGrGmCmG(6GLY)mCmGfCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT +63 RB528rGmGmGmAmGmGmAfCGrGmCmG(6GLY)mCmGfCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT +63 RB531rGmGmGmAmGmGAfCGrGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCmCiT +++63 RB532rGmGmGmAmGmGAfCGrGmCmG(6GLY)mCmGfCfCfUmGmGfCmGfUmAmAmGmCmCmUmCmCmCiT +++71 RB533rGmGmGmAmGmGAfCGrGmCmG(6GLY)mCmGfCfCfUfUmGfCmAfUmAmAmGmCmCmUmCmCmCiT +++68 RB534rGmGmGmAmGmGAfCGrGmCmG(6GLY)mCmGfCfCfUfUmGfCmGfUmAmAmGmCmCmUmCmCmCiT +++72 RB535rGmGmGmAmGmGAfCGrAmCmG(6GLY)mCmGfUfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT +++73 RB536rGmGmGmAmGmGAfCmGrAmCmG(6GLY)mCmGfUfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCmCiT +73 RB537rGmGmAmGmGAfCGrGmCmG(6GLY)mCmGfCfCfUrGmGfCmAfUmAmAmGmCmCmUmCmCiT ++++ 69RB538 rGmGmAmGmGAfCGrGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT+++ 69 RB540rGmGmAmGmGAfCGGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT +++ 69RB541 mGmGmAmGmGAfCGrGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiTND 69 (C6L) RB542mGmGmAmGmGAfCGGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT +++ 69(C6L) RB546rGmGmAmGmG(s)AfCGGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT +++69 RB547rGmGmAmGmGA(s)fCGGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT +++69 RB548rGmGmAmGmGAfC(s)GGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT +++69 RB549rGmGmAmGmGAfCG(s)GmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT ++++69 RB550rGmGmAmGmGAfCGG(s)mCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT ++++69 RB551rGmGmAmGmGAfCrGGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT +++ 69RB552rGmGmAmGmG(s)AfCrGGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT +++69 RB553rGmGmAmGmGA(s)fCrGGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT +++69 RB554rGmGmAmGmGAfC(s)rGGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT ++++69 RB555rGmGmAmGmGAfCrG(s)GmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT ++++69 RB556rGmGmAmGmGAfCrGG(s)mCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT ++++69 RB560(C6L)mGmGmAmGmGAfCG(s)GmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT++++ 69 RB561rGmGmAmGmGAfC(s)G(s)GmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT++++ 69 RB562(C6L)mGmGmAmGmGAfC(s)G(s)GmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUm ND69 CmCiT RB566rGmGmAmGmGAfCG(s)G(s)mCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT++++ 69 RB567(C6L)mGmGmAmGmGAfCG(s)G(s)mCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUm++++ 69 CmCiT RB569 (PEG4 OKGL2- ++++ 69NOF)(C6L)mGmGmAmGmGAfCG(s)GmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT RB570 (PEG4 OKGL2- +++ 69NOF)(C6L)mGmGmAmGmGAfCGGmCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiTRB571 (PEG4 OKGL2- ++++ 69NOF)(C6L)mGmGmAmGmGAfCG(s)G(s)mCmG(6GLY)mCmGfCfCfUmGmGfCmAfUmAmAmGmCmCmUmCmCiT ++++ K_(d) less than 10 nM +++ K_(d) 10 nM-24 nM ++ K_(d) 25-40nM + K_(d)>40 nM dead No Binding ND Not Determined (**) Compound assayedfunctionally All ligands described in the column titled, “ModifiedSequence” are modified versions of SEQ ID NO: 62 (RB448) rG=2’Ribo G;rA=2’Ribo A; mG=2’O-Methyl G; mA=2’O-Methyl A; mC=2’O-Methyl C;mU=2’O-Methyl U; fC=2’Fluoro C; fU=2’Fluoro U; G=2’Deoxy G; A=2’Deoxy A;iT-inverted deoxythymidine; (s)-phosphorothioate linkage;(C6L)=hexylamino linker; (6GLY)=hexaethylene glycol linker((incorporated using 9-O-Dimethoxytrityl-triethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite);(PEG40KGL2-NOF)=40 kDa Branched PEG (SUNBRIGHT GL2-400GS2 product);(6FAM): 6-carboxyfluorescein The “RB ID” is a unique identifier thatrefers to the ligand having the sequence with specific modificationsnoted in the column titled, “Modified Sequence.” The “SEQ ID NO:” refersto the corresponding nucleic acid sequence (DNA and/or RNA) withoutmodifications.

Example 5 Methods for Evaluating Antiplatelet Activity, Specificity ofActivity, and Modulation of Activity of Anti-GPVI Ligands

A. Collagen-Induced Platelet Aggregation (CIPA) Assay in PRP and WP

1. Platelet Rich Plasma Preparation (PRP) and Aggregation Studies:

Human platelet-rich plasma (PRP) was prepared from fresh whole bloodcollected in 60 ml syringes using 0.3 mM PPACK in saline (9:1blood:anticoagulant saline mix; Biomol Cat# PI1117) as an anticoagulant.The blood was centrifuged at low speed centrifugation (250×g) in 50 mlconical tubes for 16 minutes. The platelet rich plasma separated fromthe blood cells by centrifugation was removed using 10 ml serologicalpipettes and platelet poor plasma (PPP) was prepared from leftover bloodby high speed centrifugation at 2200×g for 10 minutes. The PPP wasremoved and saved for the light transmission aggregometry (LTA) blank.

Platelet aggregation in PRP was monitored using 450 μL of PRP (plus 25μL of Saline) at 37° C. (stirred at 1200 rpm) in a Chrono-Log(Havertown, Pa.) lumi-aggregometer for 6 minutes. Aggregation isinitiated using 25 μl of collagen as agonist. 500 μL of platelet-poorplasma (PPP) is used as baseline in the aggregometer. For screeninganti-GPVI ligands for the ability to block CIPA, 450 μL of PRP isincubated with 25 μL of solution containing the anti-GPVI ligand, at aconcentration to yield the desired final concentration, for 3 minutes at37° C. in the aggregometer cell with constant stirring at 1200 rpmbefore addition of the agonist collagen. Platelet aggregation isinitiated by the addition of indicated concentrations of collagen(Equine Tendon Collagen Fibril Type-1; Chronolog, Cat#385) to yield apercent aggregation between 70-90%, and the light transmission iscontinuously recorded for 4-6 min.

2. Washed Platelet Preparation (WP) and Aggregation Studies:

Human washed platelets were prepared essentially as described by Mustardet al. (1972; Br. J. Haematol 22, 193-204). Briefly, human blood wascollected into one-sixth volume of acid/citrate/dextrose (ACD) buffer(85 mM sodium citrate, 65 mM citric acid, and 110 mM glucose), placed ina water bath at 37° C. for 30 minutes then centrifuged at 250×g for 16minutes at room temperature. Platelet-rich plasma was removed andcentrifuged at 2200×g for 13 minutes at room temperature thenresuspended in 40 mL of HEPES-buffered Tyrode's solution (136.5 mM NaCl,2.68 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 12 mM NaHCO₃, 0.43 mM NaH₂PO₄, 5.5mM glucose, 5 mM HEPES pH 7.4, 0.35% bovine serum albumin) containing 10U/mL heparin and 5 μM (final concentration) prostaglandin I₂ (PGI₂). Theplatelet suspension was incubated in a 37° C. water bath for 10 minutes,5 μM (final concentration) PGI₂ added and the mixture centrifuged at1900×g for 8 minutes. The resulting pellet was resuspended in 40 mL ofHEPES-buffered Tyrode's solution containing 5 μM (final concentration)PGI₂ and then incubated for 10 minutes in a 37° C. water bath, andcentrifuged at 1900×g for 8 minutes. The pellet is resuspended at adensity of 3×10⁸ platelets/mL in HEPES-buffered Tyrode's solutioncontaining 0.1 U/mL potato apyrase and incubated in a 37° C. water bathfor 1 hr prior to use in aggregometry studies.

Collagen-induced WP platelet aggregation was determined by measuring thetransmission of light through a 0.5 ml suspension of stirred (1200 rpm)washed platelets (425 ul washed platelets, 25 μl fibrinogen, 25 μl ofinhibitors or controls and 25 μl of collagen) in a lumi-aggregometer at37° C. (Chrono-Log Corp. Havertown, Pa.). The baseline of the instrumentwas set using 0.5 ml of Hepes-buffered Tyrode's solution. Prior toaggregation measurements, the platelet suspension was supplemented with1 mg/ml fibrinogen. Platelet aggregation was initiated by the additionof indicated concentrations of Collagen (Equine Tendon Collagen FibrilType-1; Chronolog, Cat#385) to yield a percent aggregation between70-90%, and the light transmission was continuously recorded for atleast 6 min. For screening anti-GPVI ligands or controls (variousmutants of ligands) for the ability to block CIPA, anti-GPVI ligandswere added to the platelet suspension at a concentration to yield thedesired final concentration, and incubated for 3 min before addition ofcollagen, and the response was recorded for 4-6 min after collagenaddition.

The potency of collagen was determined for each donor from the maximalextent of percentage aggregation obtained from a dose response curveusing 2× serial dilution of 4 μg/ml of collagen in saline, and achallenge concentration was determined. The ability of anti-GPVI ligandsto inhibit CIPA was tested in both WP and PRP preparations as describedabove, using a broad range of anti-GPVI ligand concentrations (2 μM-7.8nM).

B. CRP Induced Platelet Aggregation (CRPIPA) Assay in WP and PRP

Cross-linked collagen related peptide, CRP-XL [(GPO)₁₀], is a selective,potent agonist of GPVI (Farndale et al., J. Thromb Haemost 2004; 2:561-573; Smethurst P A et al., J. Biol. Chem. 2007; 282: 1296-1304)which can be used to specifically trigger platelet aggregation via theGPVI receptor in both PRP and WP aggregation assays.

The potency of CRP-XL was determined for each donor from the maximalextent of percentage aggregation obtained from a dose response curveusing 2× serial dilution of 400 ng/ml of CRP-XL in saline, and achallenge concentration was determined. The ability of anti-GPVI ligandsto inhibit CRPIPA was tested in both WP and PRP preparations asdescribed above, using a broad range of anti-GPVI ligand concentrations(2 μM-7.8 nM).

C. Percent Reduction of Collagen or CRP-Induced Platelet Aggregation andIC₅₀ Determination

Percent reduction of collagen or CRP induced platelet aggregation byanti-GPVI ligands was calculated taking the maximum extent of plateletaggregation of given challenge collagen or CRP concentration as 100percent and plotted using GraphPad Prism.

When a broad range of concentrations of anti-GPVI ligands were tested(usually from 1 nM to 1 uM concentration), an IC₅₀ value was alsoobtained. IC₅₀ values represent the concentration of ligand necessary toinhibit by 50% the aggregation elicited by a given concentration ofcollagen or CRP.

D. In Vitro Flow Based Platelet Adhesion Assay in Whole Blood forAnti-GPVI Ligand Activity Using Bioflux™ 200 (Fluxion Biosciences, Inc.)

1. Preparation of the Test Plate with Collagen Coating:

For the flow experiments, Bioflux 48 well plates (P/N 0009-0013) wereroutinely used. The plates were primed with 0.02M acetic acid for 5 minat 5 dyn/cm², and then 25 μg/ml of diluted fibrillar collagen(Chrono-Log P/N 385) in 0.02M acetic acid was perfused from the inletwell for 10 min at 5 dyn/cm². The flow was stopped and the plateincubated at room temp for 1 hour. The collagen was washed with PBS at 5dyn/cm² for 10 min. The collagen coated plate was then blocked bycompletely filling the outlet well (1 ml) with PBS+5% BSA w/v andperfusing the solution into the channel at 5 dyn/cm² for 15 min. Theflow was stopped and the plate was incubated for an additional 10minutes at room temp. Excess PBS+BSA was removed from all wells and theplate was kept at room temperature for same day use or kept at 4° C. inPBS+BSA (up to two weeks).

2. Whole Blood Preparation for Perfusion and the Flow Experiment:

The blood was drawn from healthy volunteers into PPACK (0.3 mM)/CTI (60μg/ml) anticoagulant into 60 mL syringes using a 19^(3/4)″ gauge needle.The blood was immediately fluorescently labeled with 4 μM Calcein-AM(Invitrogen P/N C3100 MP) for 1 hr at 37° C. (Calcein-AM was added tothe blood very gently by inverting the tube a few times to mix and theblood was used within 3.5 hrs of draw). The experiment was initiated byadding 200 μL of labeled blood on top of the outlet well and perfusionbegun immediately using 20 dyn/cm² whole blood flow settings at 37° C.using Bioflux™ software. The data (fluorescence images of plateletaggregates) was collected using a time lapse fluorescence invertedmicroscope (Zeiss 200M Axiovert Microscope attached to an AxiocamCharged-Coupled Device camera and Axiovision software) every 6 secondsfor a total duration of 6 minutes. For test articles (anti-GPVI ligands,control ligands or control antibodies) 200 μL of labeled blood wasincubated with the indicated concentrations of ligands (or buffer-F; in10 μL volume) for 4 minutes at room temperature before addition to theoutlet well. The tagged image file (tiff) formatted images were used tocalculate fluorescence intensity using Bioflux Montage™ software andthen the data was exported to Microsoft excel and plotted using GraphpadPrism. Data was normalized to the fluorescent signal observed in thecontrol chamber at the time at which the control chamber was occluded byfluorescent platelet aggregates (defined as the maximum plateletresponse), which typically occurred between 3-4 minutes in the absenceof an anti-GPVI ligand.

E. ADP Induced Platelet Aggregation (AIPA) Assay, TRAP Induced PlateletAggregation (TIPA), Arachidonic Acid Induced Platelet Aggregation(AAIPA) Assay, and Ristocetin Induced Platelet Aggregation (RIPA) Assayfor Evaluation of Anti-GPVI Ligand Specificity for GPVI

The specificity of the anti-GPVI ligands for GPVI was determined byassessing their effect on induction of platelet aggregation by agonistswhose function is mediated through other well-characterized plateletreceptors. For these studies, human PRP and WP preparations were used asneeded to assess the activity of the various agonists. ADP was used as aspecific agonist of the P2Y₁₂ and P2Y₁ receptors, TRAP as an agonist ofPAR-1, Arachidonic Acid as an agonist of the thromboxane A2 (TXA2)receptor and Ristocetin as an agonist of the vWF-GP1bα interaction. Thepotency of each agonist (ADP, TRAP, Arachidonic Acid, and Ristocetin) isdetermined from the maximal extent of percentage aggregation obtainedfrom a dose response curve for the respective agonist, and a challengeconcentration is determined to target EC₇₀₋₉₀% for each respectiveagonist.

1. Specificity Determination of the Anti-GPVI Nucleic Acid Ligands byAIPA and TIPA in WP.

For evaluating the potential interaction of anti-GPVI ligands with P2Y₁₂and P2Y₁, a challenge concentration of 5 μM ADP was typically used tostimulate platelet aggregation, and for evaluating their potentialinteraction with PAR-1, a challenge concentration of 2.5 μM TFLLRN(TRAP) was typically used to stimulate platelet aggregation (thespecific challenge agonist concentration for each experiment isdetermined based on the agonist dose response curve for each donor). ADPand TRAP-induced platelet aggregation was determined in WP preparationsas described above. Specific inhibitors for each receptor were used aspositive controls to demonstrate that inhibition of the target receptorwas detectable in the assays. SCH79797 (Tocris Biosciences) was used asthe positive control for PAR-1 antagonism, and INS50589 (InspirePharmaceuticals) was used as the positive control for P2Y₁₂ antagonism.

2. Specificity Determination of the GPVI Nucleic Acid Ligands by RIPA inPRP:

For evaluating the potential interaction of anti-GPVI ligands with vWFor GP1bα, a challenge concentration of 1.0-2.0 mg/mL Ristocetin (SigmaCat# R7752) was used to stimulate platelet aggregation (the specificchallenge agonist concentration for each experiment is determined basedon the agonist dose response curve for each donor). Ristocetin-inducedplatelet aggregation is determined in PRP preparations as describedabove. HIP1 antibody to GP1bα (or isotype IgG control, Axxora Bio; 25μg/ml final) was used as a positive control to demonstrate thatinhibition of the target receptor GP1bα was detectable in the assays.

3. Specificity Determination of the GPVI Nucleic Acid Ligands by AAIPAin PRP:

For evaluating the potential for interaction of the anti-GPVI ligandswith TXA2 receptor, a challenge concentration of 0.25-0.5 mg/mlArachidonic Acid (Helena Biosciences; Cat#5364) was used to stimulateplatelet aggregation (the specific challenge agonist concentration foreach experiment is determined based on the agonist dose response curvefor each donor). Arachidonic Acid-induced platelet aggregation isdetermined in PRP preparations as described above.

F. Collagen and CRP-Induced Platelet Aggregation Assays for Testing ofNucleic Acid Modulators of Anti-GPVI Ligands in WP and PRP:

Collagen and CRP-induced platelet aggregation was carried out in WP andPRP preparations as described above. For evaluation of the ability ofnucleic acid modulators to reverse the inhibition of plateletaggregation by anti-GPVI ligands, anti-GPVI ligand concentration weretested at their IC₉₅₋₁₀₀ (the ligand concentration necessary to inhibitby 95-100% the aggregation elicited by a given concentration ofchallenge agonist). Platelet aggregation studies were performed asdescribed above, except that after initial incubation of the plateletpreparation with the anti-GPVI ligand, varying amounts of modulator wereadded, targeting a molar excess of modulator to ligand ranging from 8:1to 0.5:1, and incubated together for 10 minutes before addition ofagonist.

G. Durability Study of Anti-GPVI Ligand Reversal by Nucleic AcidModulators in WP:

For evaluating the durability of reversal of anti-GPVI activity bynucleic acid modulators, RB490 (final concentration 0.25 μM) and RB515(final concentration 0.75 μM) were added to a total volume 4 ml of WPsuspension at 37° C. (order and timing of addition as described abovefor evaluating nucleic acid modulators), 450 μL aliquots of the WPsuspension mixture were removed at indicated time points (0, 0.16, 0.5,1, 1.5, 2, 2.5, 3, and 3.5 hrs), and collagen-induced plateletaggregation performed. To demonstrate the activity of the WP suspension,activity of RB490, and lack of interference by RB515 over the durationof this incubation, separate incubations were conducted over the 3.5 hrtime period in which buffer alone, RB490 alone or RB515 alone were addedto WP suspensions, and collagen-induced platelet aggregation determined.

Example 6 Inhibition of Collagen and CRP-Induced Platelet Function byAnti-GPVI Ligands

GPVI, expressed exclusively on platelets, is the major platelet collagenreceptor. GPVI is required for stable platelet adhesion, and theinteraction of GPVI with collagen is one of the most potent activatorsof platelets, triggering both inside-out activation of the plateletintegrins α₂β₁ and α_(IIb)β₃, as well as secretion of platelet granulecontents (which in turn activates nearby circulating platelets). Inhumans, deficiency of GPVI causes a loss of platelet activation inresponse to collagen, and in vitro, a loss of platelet aggregation inresponse to collagen. The predicted structure of the GPVI extracellulardomain includes two Ig-like domains comprising the collagen-bindingdomain (CBD), followed by a heavily O-glycosylated stalk. Typical amongLRC receptors, GPVI associates with the FcR γ-chain co-receptor. GPVIsignaling is mediated indirectly through the γ-chain of FcR and directlythrough the GPVI cytoplasmic domain. The quaternary structure of fibrouscollagen is required for GPVI activation, and collagen-mediatedactivation can be recapitulated by the cross-linked collagen-relatedpeptide (CRP-XL), which consists of tripeptide repeats of the sequence(GPO)_(n), where G is glycine, P is proline, and O is hydroxyproline.

The crystal structure of the CBD of GPVI, consisting of residuesQ1-T183, has been solved (Horii et al, Blood 108; 2006, p936-942).Structural data along with mutagenesis data, provides detailed insightinto the molecular interaction between collagen and GPVI, and betweenCRP-XL and GPVI. Such studies also facilitate understanding of themechanisms of GPVI activation. The CBD domain consists of the twoIg-like domains, C2-1 and C2-2 (where C2-1 is N-terminal and C2-2C-terminal), oriented 90° apart. The GPVI CBD formed a dimer in thecrystal structure with the C2-2 domains of respective CBDs interactingto form a back-to-back dimer. CRP and collagen each bind, in part,within a shallow groove on C2-1. This groove is unique to GPVI among LRCreceptors, resulting from an 11-residue deletion in GPVI as compared toother LRC receptors. The floor of this binding groove is formed byseveral hydrophobic residues (L53, F54, P56, L62, and Y66 and thealiphatic portion of K41), with several polar (S43, S44, Q48, Q50, andS61) and basic (K41, R46, K59 and R166) residues around the periphery.Residues implicated directly in collagen or CRP binding fall into twoclusters. The primary region is composed of basic residues on thesurface of C2-1, including K41, K59, R60 and R166. The second cluster ofresidues implicated in CRP or collagen binding are located at the distalend of C2-1, and include L36 (implicated in collagen but not CRPbinding), and V34 and the N-glycan attached to N72, which are implicatedin both collagen and CRP binding. Consistent with mutational analysis,computational docking of CRP to the CBD of GPVI shows the CRP bindinggroove is located within the primary cluster of basic residues, withdirect interaction with K41 and R166, and within bonding distance of theside chains of K59 and R60.

Each GPVI dimer contains two collagen binding grooves, one within eachC2-1 subunit of the respective CBDs. Native collagen fibers are composedof a pseudo-hexagonal array of parallel CRP-like triple helicesseparated by 1.3 to 1.4 nm, an arrangement conserved in crystalstructures of soluble CRP peptides. The collagen binding-grooves withina GPVI dimer are essentially parallel and separated by approximately 5.5nm, the distance between n and n+4 helices in a collagen fiber. Thegeometric compatibility of the binding grooves with collagen heliceswould allow the GPVI dimer to bind simultaneously to 2 helices within acollagen fiber, and similarly to CRP-XL. Thus, the geometry of theCRP-XL peptides mimics that of native collagen, and enables the use ofCRP-XL as a discrete probe of the CBD of GPVI.

To initially assess the ability of anti-GPVI ligands to block GPVIfunction, ligands EF-2 T2 and EF-3 T2 (See FIG. 9) were evaluated fortheir ability to block collagen-induced platelet activation in WPpreparations, as described in Example 5. As a control for any potentialnon-specific effects of ligands on collagen-induced plateletaggregation, non-GPVI binding mutant ligands, EF-2 T2 M3-M5 and EF-3 T2M3-M5 (See FIG. 9) were included in initial screens as a specificitycontrol. As shown in FIG. 15, both EF-2 T2 and EF-3 T2 effectivelyblocked collagen-induced platelet aggregation, whereas inactive mutantcontrol ligands had no effect.

Subsequently, the activity of optimized anti-GPVI ligands RB490, RB540,RB549 and RB566 were evaluated in collagen-induced platelet aggregationin WP preparations (FIG. 16). Optimized ligands each effectively blockedcollagen-induced platelet aggregation. The relative potencies' of theanti-GPVI ligands were consistent with their respective affinities forGPVI, with RB540 (K_(d) for GPVI of ˜15 nM) exhibiting an IC₅₀ of ˜44nM, RB549 (K_(d) for GPVI of ˜6-7 nM) exhibiting an IC₅₀ of ˜25 nM,RB490 (K_(d) for GPVI of ˜4-5 nM) exhibiting an IC₅₀ of ˜23 nM, andRB566 (K_(d) for GPVI of ˜2-3 nM) exhibiting an IC₅₀ of ˜20 nM.

To further probe the potential mechanism by which the ant-GPVI ligandsblock GPVI function, ligands RB490, RB549 and RB566 were evaluated fortheir ability to block CRP-XL induced platelet aggregation in WPpreparations (FIG. 17), as described in Example 5. Consistent with theirability to block collagen-induced platelet aggregation, RB490, RB549 andRB566 blocked CRP-induced platelet aggregation in a dose-dependentmanner. As with collagen, the relative potency of the ligands wasconsistent with their respective affinities for GPVI, with IC₅₀'s of 38nM, 79 nM and 98 nM for RB566, RB490 and RB549, respectively. Notsurprisingly, the IC₅₀'s of the anti-GPVI ligands are higher inCRP-induced platelet aggregation, as the CRP-XL used in these assays isa more potent platelet agonist than collagen. Together, these data areconsistent with a mechanism of action in which the anti-GPVI ligandsbind GPVI at or near the collagen-binding groove of GPVI, therebyblocking association of collagen or CRP-XL with GPVI on the plateletsurface.

The anti-GPVI activity of PEGylated ligands RB570, RB569 and RB571 wasalso compared to RB490 in collagen and CRP-induced platelet aggregationin WP preparations (FIGS. 18-19). As above, the PEGylated ligandsexhibited similar dose-dependent inhibition of collagen and CRP-inducedplatelet aggregation. The addition of PEG to the ligands modestlyimpacted their activity relative to RB490 in collagen-induced plateletaggregation, but not in CRP-induced platelet aggregation. RB571exhibited the greatest potency in blocking CRP-induced aggregation in WPpreparations, with an IC₅₀ of ˜33 nM. Likewise, RB571 exhibited potentinhibition of collagen and CRP-induced platelet aggregation in PRP (SeeFIG. 20; See Example 5 for details).

Anti-GPVI ligands were also evaluated for their ability to blockplatelet adhesion, activation and aggregation in response to GPVIinteraction with collagen coated surfaces in whole blood under highshear, as described in Example 5. The format of this assay reflects manyfeatures of the in vivo function of GPVI, including association of GPVIwith surface bound fibrillar collagen in flowing whole blood, as well asrecapitulating three critical functions mediated by the GPVI-collageninteraction, namely platelet adhesion, activation and aggregation. RB566and RB571 greatly reduced platelet adhesion, activation and aggregation(FIG. 21), as compared to the inactive ligand control RB471 (plateletresponse in this assay was comparable between RB471 and buffer control).

Thus, anti-GPVI ligands block GPVI function in isolated platelet assaysunder static conditions and in flowing whole blood, impactingGPVI-mediated platelet adhesion, activation and aggregation.Additionally, consistent with a mechanism in which the anti-GPVI ligandsbind at or near to the collagen binding site on GPVI, they exhibitdose-dependent inhibition of both collagen and CRP-induced plateletaggregation with a relative potency consistent with their respectiveaffinities for GPVI.

Example 7 Specificity of Anti-GPVI Ligands for GPVI

GPVI is one of several platelet receptors via which activation by itscognate ligand stimulates platelet activation and aggregation. Othersuch receptors include the thromboxane A2 receptor (TXA2), the ADPreceptors P2Y₁₂ and P2Y₁, the thrombin receptor PAR-I, and GP1bα, whichis activated by collagen-bound von Willebrands Factor (vWF). Todetermine the specificity with which the anti-GPVI ligands bind GPVI andblock platelet function, the effect of the anti-GPVI ligand RB571 wasassessed in ADP, TRAP (PAR-1 agonist), Arachidonic Acid (TXA2 agonist)and Ristocetin (agonist of the vWF-GP1bα interaction) induced plateletaggregation assays, as described in Example 5. As shown in FIGS. 22 and23, RB571 had no impact on ADP or TRAP-induced platelet aggregation inWP preparations, or on Ristocetin or Arachidonic Acid-induced plateletaggregation in PRP preparations. Thus, as expected based on their highaffinity for GPVI and isolation via SELEX, the anti-GPVI ligands arespecific for GPVI.

Example 8 Nucleic Acid Modulators of Anti-GPVI Ligands

Ligands encode the information necessary to design nucleic acidmodulators, or control agents, for them based upon complementaryWatson-Crick basepairing rules. The effectiveness of a given controlagent is dependent upon several factors, including accessibility of thetargeted region of the ligand for nucleation with the control agent, aswell as the absence of or limited internal secondary structure withinthe control agent, which would require denaturation prior to full-duplexformation with the ligand. To define regions of the anti-GPVI ligandsthat would be preferred regions for association with nucleic acidmodulators, a series of control agents (See Table 7 and FIG. 24) weredesigned for EF-2 T2 and EF-3 T2.

Table 7: Modulators of GPVI Ligands

TABLE 7 Modulators of GPVI Ligands SEQ ID Name NO: RB IDModified Sequence EF-2 CA1 74 RB416 mAmGmGmAmGmGmCmUmUmAmUmGmCmAmAmGEF-2 CA2 75 RB417 mGmAmGmGmCmUmUmAmUmGmCmAmAmGmAmCmG EF-2 CA3 76 RB418mUmUmAmUmGmCmAmAmGmAmCmGmGmGmUmG EF-2 CA4 77 RB419mUmUmUmUmGmGmCmAmCmCmGmCmAmUmCmGmUmC EF-3 CA1 78 RB420mAmGmGmAmGmGmCmUmUmAmCmGmCmAmAmG EF-3 CA2 79 RB421mGmAmGmGmCmUmUmAmCmGmCmAmAmGmAmCmG EF-3 CA3 80 RB422mUmUmAmCmGmCmAmAmGmAmCmGmCmGmGmU EF-3 CA4 81 RB423mUmGmUmGmAmUmCmCmGmCmAmUmCmGmUmC RB490 CA 1 82 RB513mGmGmGmAmGmGmCmUmUmAmUmGmCmCmAmGmGmCmG RB490 CA 2 83 RB514mGmAmGmGmCmUmUmAmUmGmCmCmAmGmGmCmG RB490 CA 3 84 RB515mUmUmAmUmGmCmCmAmGmGmCmG RB490 CA 4 85 RB516 mCmGmCmCmGmUmCmCmUmCmCmCRB538/571 86 RB543 mGmCmUmUmAmUmGmCmCmAmGmGmCmG Control Agent 5 (14 mer)RB538/571 87 RB544 mGmGmCmUmUmAmUmGmCmCmAmGmGmCmG Control Agent 6(15 mer) RB538/571 88 RB545 mAmGmGmCmUmUmAmUmGmCmCmAmGmGmCmGControl Agent 7 (16 mer) SEQ ID NOs. 137-151 correspond to theunmodified versions of the modulators described in the column titled“Modified Sequence.”

The reversal activity of control agents RB416-RB419 for EF-2 T2 andRB420-423 for EF-3 T2 was assessed in platelet aggregation assays, asdescribed in Example 5. As shown in FIGS. 25 and 26, the control agentswere capable of reversing anti-GPVI ligand function within 10 minutes atlow molar excesses to the GPVI ligand. A consistent potency between thecontrol agents designed for EF-2 T2 and EF-3 T2 was observed withrespect to the complementary regions of the control agents on theanti-GPVI ligands. RB418 and RB422, which associate with a similarregion of the anti-GPVI ligands consisting of the 3′ side of Stem 2,Loop 3, the 5′ side of Stem 3 and Loop 4, exhibited the greatestpotency. These control agents are expected to exhibit similar potencyprofiles to any of the described anti-GPVI ligands with which they canform fully-complementary duplexes.

A similar set of potential control agents was designed against RB490(See Table 7). As shown in FIG. 27, the potency with which these nucleicacid modulators reversed the anti-GPVI activity of RB490 was consistentwith the preferred pairing regions as defined for control agents toEF-2T and EF-3T, with RB515 exhibiting the greatest potency. A keyfeature of a modulator is that once complexed with the ligand, themodulator:ligand complex does not dissociate, and the ligand activityremains durably reversed. To assess the durability of reversal of RB490by RB515, we measured the durability of reversal of the anti-GPVIactivity of RB490 in collagen-induced platelet aggregation, as describedin Example 5. RB515 durably reversed RB490 activity for 3.5 hrs (FIG.28), the duration in which the WP preparations maintained fullaggregation activity in response to collagen. Notably, RB490 in theabsence of RB515 continued to fully inhibit collagen-induced plateletaggregation over the course of the 3.5 hr incubation period. Theduration of reversal activity of RB515 for the anti-GPVI activity ofRB490 is comparable to that determined for reversal of the anticoagulantactivity of the anti-FIXa ligand RB006 with control agent RB007 in vitroin human plasma, an ligand:control agent pair which has demonstrateddurable reversal in vivo in humans as well as other species.

RB571 and RB490 share the same base sequence within the region to whichRB515 is complementary, and thus RB515 is a potential control agent forRB571 (See FIG. 29). Therefore, the reversal activity of RB515 for RB571was assessed in collagen and CRP-induced platelet activation in WP andPRP preparations. RB515, at a low molar excess, effectively reversed theanti-GPVI activity of RB571 in response to both agonists in bothmatrices (FIGS. 30 and 31). The molar excess required for completereversal of RB571 activity by RB515 is less than that required for fullreversal of RB006 by RB007 in vitro (2:1 or lower molar ratio ascompared to 4:1 for RB007 reversal of RB006), indicating a highlikelihood of success for reversal of RB571 by RB515 in vivo in humansand other species.

The invention claimed is:
 1. A nucleic acid modulator which bindsspecifically to a GPVI ligand, wherein said GPVI ligand is a nucleicacid that comprises a ligand nucleic acid sequence, wherein the ligandnucleic acid sequence is characterized by a secondary structurecomprising in a 5′ to 3′ direction a first stem, a first loop, a secondstem, a second loop, a third loop, a third stem and a fourth loop; andwherein said fourth loop comprises a consensus sequence comprising UAA.2. The modulator of claim 1, wherein the ligand nucleic acid sequenceconsists of about 10 nt to about 50 nt.
 3. The modulator of claim 1comprising SEQ ID NO:84.
 4. The modulator of claim 3, wherein themodulator is RB515 of Table
 7. 5. The modulator of claim 1, wherein themodulator comprises a modulator nucleic acid sequence comprising3′-AUU-5′ and which is capable of binding specifically to the fourthloop of the GPVI nucleic acid ligand.
 6. The modulator of claim 1,wherein the modulator comprises a modulator nucleic acid sequence thatis complementary to at least the first loop, the second stem, and thesecond loop of the nucleic acid GPVI ligand.
 7. The modulator of claim1, wherein the modulator comprises a modulator nucleic acid sequencecomprising 3′-CUG-5′ and the modulator is capable of bindingspecifically to the first loop of the GPVI nucleic acid ligand.
 8. Themodulator of claim 1, wherein the modulator reverses, partially orcompletely, the activity of the GPVI ligand when bound to the GPVIligand.
 9. The modulator of claim 1, wherein the ligand nucleic acidsequence comprises at least one modified nucleotide.
 10. The modulatorof claim 9, wherein the at least one modified nucleotide is selectedfrom 2′-O-methyl and 2′-fluoro.
 11. The modulator of claim 10, whereinthe at least one modified nucleotide is selected from 2′-O-methylcytosine, 2′-O-methyl uridine, 2′-O-methyl adenosine, 2′-O-methylguanosine or a 2′-O-methyl thymidine.
 12. The modulator of claim 10,wherein the at least one modified nucleotide is selected from 2′ fluorocytidine, a 2′ fluoro uridine, a 2′ fluoro adenosine or a 2′-fluoroguanosine.
 13. The modulator of claim 1, further comprising a carrier.14. The modulator of claim 13, wherein the carrier is a hydrophilicmoiety.
 15. The modulator of claim 14, wherein the hydrophilic moiety ispolyethylene glycol (PEG) molecule.
 16. The modulator of claim 1,wherein the modulator comprises a modified phosphate backbone.
 17. Themodulator of claim 1, wherein the modulator comprises as least onemodified sugar moiety.
 18. The modulator of claim 1, wherein themodulator is selected from RB416, RB417, RB418, RB419, RB420, RB421,RB422, RB423, RB513, RB514, RB515, RB516, RB543, RB544, or RB545 ofTable
 7. 19. The modulator of claim 1, wherein the binding of themodulator to the GPVI ligand exposes a suicide position within the GPVIligand, thereby disrupting the secondary structure of the GPVI ligandand leading to enhanced destruction of the nucleic acid GPVI ligand bynucleases.
 20. A pharmaceutical composition comprising the modulator ofclaim
 1. 21. A pharmaceutical composition comprising the modulator ofclaim 4.